技术资料

Ethanol (EtOH)-induced alterations in intestinal homeostasis lead to multi-system pathologies,including liver injury. $\omega$-6 PUFAs exert pro-inflammatory activity,while $\omega$-3 PUFAs promote anti-inflammatory activity that is mediated,in part,through specialized pro-resolving mediators [e.g.,resolvin D1 (RvD1)]. We tested the hypothesis that a decrease in the $\omega$-6:$\omega$-3 PUFA ratio would attenuate EtOH-mediated alterations in the gut-liver axis. $\omega$-3 FA desaturase-1 (fat-1) mice,which endogenously increase $\omega$-3 PUFA levels,were protected against EtOH-mediated downregulation of intestinal tight junction proteins in organoid cultures and in vivo. EtOH- and lipopolysaccharide-induced expression of INF-$\gamma$,Il-6,and Cxcl1 was attenuated in fat-1 and WT RvD1-treated mice. RNA-seq of ileum tissue revealed upregulation of several genes involved in cell proliferation,stem cell renewal,and antimicrobial defense (including Alpi and Leap2) in fat-1 versus WT mice fed EtOH. fat-1 mice were also resistant to EtOH-mediated downregulation of genes important for xenobiotic/bile acid detoxification. Further,gut microbiome and plasma metabolomics revealed several changes in fat-1 versus WT mice that may contribute to a reduced inflammatory response. Finally,these data correlated with a significant reduction in liver injury. Our study suggests that $\omega$-3 PUFA enrichment or treatment with resolvins can attenuate the disruption in intestinal homeostasis caused by EtOH consumption and systemic inflammation with a concomitant reduction in liver injury. View Publication

Mammalian focal adhesion proteins Pinch1 and Pinch2 regulate integrin activation and cell-extracellular matrix adhesion and migration. Here,we show that deleting Pinch1 in osteocytes and mature osteoblasts using the 10-kb mouse Dmp1-Cre and Pinch2 globally (double KO; dKO) results in severe osteopenia throughout life,while ablating either gene does not cause bone loss,suggesting a functional redundancy of both factors in bone. Pinch deletion in osteocytes and mature osteoblasts generates signals that inhibit osteoblast and bone formation. Pinch-deficient osteocytes and conditioned media from dKO bone slice cultures contain abundant sclerostin protein and potently suppress osteoblast differentiation in primary BM stromal cells (BMSC) and calvarial cultures. Pinch deletion increases adiposity in the BM cavity. Primary dKO BMSC cultures display decreased osteoblastic but enhanced adipogenic,differentiation capacity. Pinch loss decreases expression of integrin $\beta$3,integrin-linked kinase (ILK),and $\alpha$-parvin and increases that of active caspase-3 and -8 in osteocytes. Pinch loss increases osteocyte apoptosis in vitro and in bone. Pinch loss upregulates expression of both Rankl and Opg in the cortical bone and does not increase osteoclast formation and bone resorption. Finally,Pinch ablation exacerbates hindlimb unloading-induced bone loss and impairs active ulna loading-stimulated bone formation. Thus,we establish a critical role of Pinch in control of bone homeostasis. View Publication

The perivascular niche within adipose tissue is known to house multipotent cells,including osteoblast precursors. However,the identity of perivascular subpopulations that may mineralize or ossify most readily is not known. Here,we utilize inducible PDGFR$\alpha$ (platelet-derived growth factor alpha) reporter animals to identify subpopulations of perivascular progenitor cells. Results showed that PDGFR$\alpha$-expressing cells are present in four histologic niches within inguinal fat,including two perivascular locations. PDGFR$\alpha$+ cells are most frequent within the tunica adventitia of arteries and veins,where PDGFR$\alpha$+ cells populate the inner aspects of the adventitial layer. Although both PDGFR$\alpha$+ and PDGFR$\alpha$- fractions are multipotent progenitor cells,adipose tissue-derived PDGFR$\alpha$+ stromal cells proliferate faster and mineralize to a greater degree than their PDGFR$\alpha$- counterparts. Likewise,PDGFR$\alpha$+ ectopic implants reconstitute the perivascular niche and ossify to a greater degree than PDGFR$\alpha$- cell fractions. Adventicytes can be further grouped into three distinct groups based on expression of PDGFR$\alpha$ and/or CD34. When further partitioned,adventicytes co-expressing PDGFR$\alpha$ and CD34 represented a cell fraction with the highest mineralization potential. Long-term tracing studies showed that PDGFR$\alpha$-expressing adventicytes give rise to adipocytes,but not to other cells within the vessel wall under homeostatic conditions. However,upon bone morphogenetic protein 2 (BMP2)-induced ossicle formation,descendants of PDGFR$\alpha$+ cells gave rise to osteoblasts,adipocytes,and pericyte-like" cells within the ossicle. In sum PDGFR$\alpha$ marks distinct perivascular osteoprogenitor cell subpopulations within adipose tissue. The identification of perivascular osteoprogenitors may contribute to our improved understanding of pathologic mineralization/ossification. Stem Cells 2019." View Publication

Molecular- and cellular-based therapies have the potential to reduce obesity-associated disease. In response to cold,beige adipocytes form in subcutaneous white adipose tissue and convert energy stored in metabolic substrates to heat,making them an attractive therapeutic target. We developed a robust method to generate a renewable source of human beige adipocytes from induced pluripotent stem cells (iPSCs). Developmentally,these cells are derived from FOXF1+ mesoderm and progress through an expandable mural-like mesenchymal stem cell (MSC) to form mature beige adipocytes that display a thermogenically active profile. This includes expression of uncoupling protein 1 (UCP1) concomitant with increased uncoupled respiration. With this method,dysfunctional adipogenic precursors can be reprogrammed and differentiated into beige adipocytes with increased thermogenic function and anti-diabetic secretion potential. This resource can be used to (1) elucidate mechanisms that underlie the control of beige adipogenesis and (2) generate material for cellular-based therapies that target metabolic syndrome in humans. View Publication

TFF1,a secreted protein,plays an essential role in keeping the integrity of gastric mucosa and its barrier function. Loss of TFF1 expression in the TFF1-knockout (KO) mouse leads to a pro-inflammatory phenotype with a cascade of gastric lesions that include low-grade dysplasia,high-grade dysplasia,and adenocarcinomas. In this study,we demonstrate nuclear localization of p-STATY705,with significant overexpression of several STAT3 target genes in gastric glands from the TFF1-KO mice. We also show frequent loss of TFF1 with nuclear localization of STAT3 in human gastric cancers. The reconstitution of TFF1 protein in human gastric cancer cells and 3D gastric glands organoids from TFF1-KO mice abrogates IL6-induced nuclear p-STAT3Y705 expression. Reconstitution of TFF1 inhibits IL6-induced STAT3 transcription activity,suppressing expression of its target genes. TFF1 blocks IL6R$\alpha$-GP130 complex formation through interfering with binding of IL6 to its receptor IL6R$\alpha$. These findings demonstrate a functional role of TFF1 in suppressing gastric tumorigenesis by impeding the IL6-STAT3 pro-inflammatory signaling axis. View Publication

Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes,including the release of proteases,phagocytosis and the production of neutrophil extracellular traps (NETs). NET formation,or NETosis,is a specific and highly efficient process,which is induced by a variety of stimuli leading to expulsion of DNA,proteases and antimicrobial peptides to the extracellular space. However,uncontrolled NETosis may lead to adverse effects and exert tissue damage in pathological conditions. Here,we show that the ATP channel pannexin1 (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). Interestingly,neutrophils isolated from Panx1-/- mice showed reduced and/or delayed induction of NETosis. Brilliant blue FCF dye (BB-FCF),a Panx1 channel inhibitor,decreased NETosis in wild-type neutrophils to the extent observed in Panx1-/- neutrophils. Thus,we demonstrate that ATP and Panx1 channels contribute to NETosis and may represent a therapeutic target. View Publication

Many adult tissues contain resident stem cells,such as the Pax7+ satellite cells within skeletal muscle,that regenerate parenchymal elements following damage. Tissue-resident mesenchymal progenitors (MPs) also participate in regeneration,although their function and fate in this process are unclear. Here,we identify Hypermethylated in cancer 1 (Hic1) as a marker of MPs in skeletal muscle and further show that Hic1 deletion leads to MP hyperplasia. Single-cell RNA-seq and ATAC-seq analysis of Hic1+ MPs in skeletal muscle shows multiple subpopulations,which we further show have distinct functions and lineage potential. Hic1+ MPs orchestrate multiple aspects of skeletal muscle regeneration by providing stage-specific immunomodulation and trophic and mechanical support. During muscle regeneration,Hic1+ derivatives directly contribute to several mesenchymal compartments including Col22a1-expressing cells within the myotendinous junction. Collectively,these findings demonstrate that HIC1 regulates MP quiescence and identifies MP subpopulations with transient and enduring roles in muscle regeneration. View Publication

Ex vivo retroviral gene transfer into CD34+ hematopoietic stem and progenitor cells (HSPCs) has demonstrated remarkable clinical success in gene therapy for monogenic hematopoietic disorders. However,little attention has been paid to enhancement of culture and transduction conditions to achieve reliable effects across patient and disease contexts and to maximize potential vector usage and reduce treatment cost. We systematically tested three HSPC culture media manufactured to cGMP and eight previously described transduction enhancers (TEs) to develop a state-of-the-art clinically applicable protocol. Six TEs enhanced lentiviral (LV) and five TEs facilitated alpharetroviral (ARV) CD34+ HSPC transduction when used alone. Combinatorial TE application tested with LV vectors yielded more potent effects,with up to a 5.6-fold increase in total expression of a reporter gene and up to a 3.8-fold increase in VCN. Application of one of the most promising combinations,the poloxamer LentiBOOST and protamine sulfate,for GMP-compliant manufacturing of a clinical-grade advanced therapy medicinal product (ATMP) increased total VCN by over 6-fold,with no major changes in global gene expression profiles or inadvertent loss of CD34+CD90+ HSPC populations. Application of these defined culture and transduction conditions is likely to significantly improve ex vivo gene therapy manufacturing protocols for HSPCs and downstream clinical efficacy. View Publication

The sodium potassium pump (Na/K-ATPase) ensures the electrochemical gradient of a cell through an energy-dependent process that consumes about one-third of regenerated ATP. We report that the G protein-coupled receptor GPR35 interacted with the $\alpha$ chain of Na/K-ATPase and promotes its ion transport and Src signaling activity in a ligand-independent manner. Deletion of Gpr35 increased baseline Ca2+ to maximal levels and reduced Src activation and overall metabolic activity in macrophages and intestinal epithelial cells (IECs). In contrast,a common T108M polymorphism in GPR35 was hypermorphic and had the opposite effects to Gpr35 deletion on Src activation and metabolic activity. The T108M polymorphism is associated with ulcerative colitis and primary sclerosing cholangitis,inflammatory diseases with a high cancer risk. GPR35 promoted homeostatic IEC turnover,whereas Gpr35 deletion or inhibition by a selective pepducin prevented inflammation-associated and spontaneous intestinal tumorigenesis in mice. Thus,GPR35 acts as a central signaling and metabolic pacesetter,which reveals an unexpected role of Na/K-ATPase in macrophage and IEC biology. View Publication

As the prognosis of invasive aspergillosis remains unacceptably poor in patients undergoing hematopoietic stem cell transplantation (HSCT),there is a growing interest in the adoptive transfer of antifungal effector cells,such as Natural Killer (NK) cells. Because immunosuppressive agents are required in most HSCT recipients,knowledge of the impact of these compounds on the antifungal activity of NK cells is a prerequisite for clinical trials. We,therefore,assessed the effect of methylprednisolone (mPRED),cyclosporin A (CsA) and mycophenolic acid (MPA) at different concentrations on proliferation,apoptosis/necrosis,and the direct and indirect anti-Aspergillus activity of human NK cells. Methylprednisolone decreased proliferation and increased apoptosis of NK cells in a significant manner. After seven days,a reduction of viable NK cells was seen for all three immunosuppressants,which was significant for MPA only. Cyclosporin A significantly inhibited the direct hyphal damage by NK cells in a dose-dependent manner. None of the immunosuppressive compounds had a major impact on the measured levels of interferon-$\gamma$,granulocyte-macrophage colony-stimulating factor and RANTES (regulated on activation,normal T cell expressed and secreted; CCL5). Our data demonstrate that commonly used immunosuppressive compounds have distinct effects on proliferation,viability and antifungal activity of human NK cells,which should be considered in designing studies on the use of NK cells for adoptive antifungal immunotherapy. View Publication

Despite advances in therapy,glioblastoma remains an incurable disease with a dismal prognosis. Recent studies have implicated cancer stem cells within glioblastoma (glioma stem cells,GSCs) as mediators of therapeutic resistance and tumor progression. In this study,we investigated the role of the transforming growth factor-$\beta$ (TGF-$\beta$) superfamily,which has been found to play an integral role in the maintenance of stem cell homeostasis within multiple stem cell systems,as a mediator of stem-like cells in glioblastoma. We find that BMP and TGF-$\beta$ signaling define divergent molecular and functional identities in glioblastoma,and mark relatively quiescent and proliferative GSCs,respectively. Treatment of GSCs with BMP inhibits cell proliferation,but does not abrogate their stem-ness,as measured by self-renewal and tumorigencity. Further,BMP pathway activation confers relative resistance to radiation and temozolomide chemotherapy. Our findings define a quiescent cancer stem cell population in glioblastoma that may be a cellular reservoir for tumor recurrence following cytotoxic therapy. View Publication

Protein disulfide isomerase (PDI,PDIA1) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to selectively induce apoptosis in cancer cells and overcome resistance to existing therapies,although drug candidates have not yet progressed to the stage of clinical development. We recently reported the discovery of lead indene compound E64FC26 as a potent pan-PDI inhibitor that enhances the cytotoxic effects of proteasome inhibitors in panels of Multiple Myeloma (MM) cells and MM mouse models. An extensive medicinal chemistry program has led to the generation of a diverse library of indene-containing molecules with varying degrees of proteasome inhibitor potentiating activity. These compounds were generated by a novel nucleophilic aromatic ring cyclization and dehydration reaction from the precursor ketones. The results provide detailed structure activity relationships (SAR) around this indene pharmacophore and show a high degree of correlation between potency of PDI inhibition and bortezomib (Btz) potentiation in MM cells. Inhibition of PDI leads to ER and oxidative stress characterized by the accumulation of misfolded poly-ubiquitinated proteins and the induction of UPR biomarkers ATF4,CHOP,and Nrf2. This work characterizes the synthesis and SAR of a new chemical class and further validates PDI as a therapeutic target in MM as a single agent and in combination with proteasome inhibitors. View Publication