技术资料

NF-$\kappa$B is a key regulator of inflammation and cancer progression,with an important role in leukemogenesis. Despite its therapeutic potential,targeting NF-$\kappa$B using pharmacologic inhibitors has proven challenging. Here,we describe a myeloid cell-selective NF-$\kappa$B inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a,C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6),thereby blocking activation of NF-$\kappa$B in target cells. IV injections of C-miR146a mimic to miR-146a-deficient mice prevented excessive NF-$\kappa$B activation in myeloid cells,and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly,C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell-induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions,miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set,we found an inverse correlation of miR-146a levels with NF-$\kappa$B-related genes and with patient survival. Correspondingly,C-miR146a induced cytotoxic effects in human MDSL,HL-60,and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-$\kappa$B target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell-targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders. View Publication

R-2-hydroxyglutarate (R-2HG),produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes,was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically,R-2HG inhibits fat mass and obesity-associated protein (FTO) activity,thereby increasing global N6-methyladenosine (m6A) RNA modification in R-2HG-sensitive leukemia cells,which in turn decreases the stability of MYC/CEBPA transcripts,leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG,whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively,while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation,our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/m6A/MYC/CEBPA signaling. View Publication

Leukocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in host defense and immune regulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD),characterized by recurrent bacterial and fungal infections and aberrant inflammation. Key drivers of hyper-inflammation induced by fungal cell walls in CGD are still incompletely defined. Here,we found that CGD (CYBB-null) neutrophils produced higher amounts of leukotriene B4 (LTB4) in vitro following activation with zymosan or Immune complexes,as compared to wild type (WT) neutrophils. This correlated with increased calcium influx in CGD neutrophils,which is restrained in WT neutrophils by the electrogenic activity of the NADPH oxidase. Increased LTB4 generation by CGD neutrophils was also augmented by paracrine cross-talk with the LTB4 receptor BLT1. CGD neutrophils formed more numerous and larger clusters in the presence of zymosan in vitro compared to WT,which was also LTB4- and BLT1-dependent. In zymosan-induced lung inflammation,focal neutrophil infiltrates were increased in CGD compared to WT mice and associated with higher LTB4 levels. Inhibiting LTB4 synthesis or antagonizing the BLT1 receptor following zymosan challenge reduced lung neutrophil recruitment in CGD to WT levels. Thus,LTB4 was the major driver of excessive neutrophilic lung inflammation in CGD mice in the early response to fungal cell walls,likely by a dysregulated feed-forward loop involving amplified neutrophil production of LTB4. This study identifies neutrophil LTB4 generation as a target of NADPH oxidase regulation,which could potentially be exploited therapeutically to reduce excessive inflammation in CGD. View Publication

Immune surveillance against pathogens and tumours in the central nervous system is thought to be limited owing to the lack of lymphatic drainage. However,the characterization of the meningeal lymphatic network has shed light on previously unappreciated ways that an immune response can be elicited to antigens that are expressed in the brain1-3. Despite progress in our understanding of the development and structure of the meningeal lymphatic system,the contribution of this network in evoking a protective antigen-specific immune response in the brain remains unclear. Here,using a mouse model of glioblastoma,we show that the meningeal lymphatic vasculature can be manipulated to mount better immune responses against brain tumours. The immunity that is mediated by CD8 T cells to the glioblastoma antigen is very limited when the tumour is confined to the central nervous system,resulting in uncontrolled tumour growth. However,ectopic expression of vascular endothelial growth factor C (VEGF-C) promotes enhanced priming of CD8 T cells in the draining deep cervical lymph nodes,migration of CD8 T cells into the tumour,rapid clearance of the glioblastoma and a long-lasting antitumour memory response. Furthermore,transfection of an mRNA construct that expresses VEGF-C works synergistically with checkpoint blockade therapy to eradicate existing glioblastoma. These results reveal the capacity of VEGF-C to promote immune surveillance of tumours,and suggest a new therapeutic approach to treat brain tumours. View Publication

Aging impairs the regenerative potential of hematopoietic stem cells (HSC) and skews differentiation towards the myeloid lineage. The bone marrow (BM) microenvironment has recently been suggested to influence HSC aging,however the mechanisms whereby BM stromal cells mediate this effect is unknown. Here we show that aging-associated decreased expression of CXCR4 expression on BM mesenchymal stem cells (MSC) plays a crucial role in the development of the hematopoietic stem and progenitor cells (HSPC) aging phenotype. The BM MSC from old mice was sufficient to drive a premature aging phenotype of young HSPC when cultured together ex vivo. The impaired ability of old MSC to support HSPC function is associated with reduced expression of CXCR4 on BM MSC of old mice. Deletion of the CXCR4 gene in young MSC accelerates an aging phenotype in these cells characterized by increased production of reactive oxygen species (ROS),DNA damage,senescence,and reduced proliferation. Culture of HSPC from young mice with CXCR4 deficient MSC also from young mice led to a premature aging phenotype in the young HSPC,as evidenced by reduced hematopoietic regeneration and enhanced myeloid differentiation. Mechanistically,CXCR4 signaling prevents BM MSC dysfunction by suppressing oxidative stress,as treatment of old or CXCR4 deficient MSC with N-acetyl-L-cysteine (NAC),improved their niche supporting activity,and attenuated the HSPC aging phenotype. Our studies suggest that age-associated reduction in CXCR4 expression on BM MSC impairs hematopoietic niche activity with increased ROS production,driving an HSC aging phenotype. Thus,modulation of the SDF-1/CXCR4 axis in MSC may lead to novel interventions to alleviate the age-associated decline in immune/hematopoietic function. View Publication

The diagnosis of Parkinson's disease (PD) is initiated after the occurrence of motor symptoms,such as resting tremors,rigidity,and bradykinesia. According to previous reports,non-motor symptoms,notably gastrointestinal dysfunction,could potentially be early biomarkers in PD patients as such symptoms occur earlier than motor symptoms. However,connecting PD to the intestine is methodologically challenging. Thus,we generated in vitro human intestinal organoids from PD patients and ex vivo mouse small intestinal organoids from aged transgenic mice. Both intestinal organoids (IOs) contained the human LRRK2 G2019S mutation,which is the most frequent genetic cause of familial and sporadic PD. By conducting comprehensive genomic comparisons with these two types of IOs,we determined that a particular gene,namely,Iroquois homeobox protein 2 (IRX2),showed PD-related expression patterns not only in human pluripotent stem cell (PSC)-derived neuroectodermal spheres but also in human PSC-derived neuronal cells containing dopaminergic neurons. We expected that our approach of using various cell types presented a novel technical method for studying the effects of multi-organs in PD pathophysiology as well as for the development of diagnostic markers for PD. View Publication

The cerebellum plays a critical role in all vertebrates,and many neurological disorders are associated with cerebellum dysfunction. A major limitation in cerebellar research has been the lack of adequate disease models. As an alternative to animal models,cerebellar neurons differentiated from pluripotent stem cells have been used. However,previous studies only produced limited amounts of Purkinje cells. Moreover,in vitro generation of Purkinje cells required co-culture systems,which may introduce unknown components to the system. Here we describe a novel differentiation strategy that uses defined medium to generate Purkinje cells,granule cells,interneurons,and deep cerebellar nuclei projection neurons,that self-formed and differentiated into electrically active cells. Using a defined basal medium optimized for neuronal cell culture,we successfully promoted the differentiation of cerebellar precursors without the need for co-culturing. We anticipate that our findings may help developing better models for the study of cerebellar dysfunctions,while providing an advance toward the development of autologous replacement strategies for treating cerebellar degenerative diseases. View Publication

Understanding metabolic pathways that regulate Th17 development is important to broaden therapeutic options for Th17-mediated autoimmunity. Here,we report a pivotal role of mitochondrial oxidative phosphorylation (OXPHOS) for lineage specification toward pathogenic Th17 differentiation. Th17 cells rapidly increase mitochondrial respiration during development,and this is necessary for metabolic reprogramming following T cell activation. Surprisingly,specific inhibition of mitochondrial ATP synthase ablates Th17 pathogenicity in a mouse model of autoimmunity by preventing Th17 pathogenic signature gene expression. Notably,cells activated under OXPHOS-inhibited Th17 conditions preferentially express Foxp3,rather than Th17 genes,and become suppressive Treg cells. Mechanistically,OXPHOS promotes the Th17 pioneer transcription factor,BATF,and facilitates T cell receptor (TCR) and mTOR signaling. Correspondingly,overexpression of BATF rescues Th17 development when ATP synthase activity is restricted. Together,our data reveal a regulatory role of mitochondrial OXPHOS in dictating the fate decision between Th17 and Treg cells by supporting early molecular events necessary for Th17 commitment. View Publication

Human pluripotent stem cells including induced pluripotent stem cells (iPSCs) and embryonic stem cells hold great promise for cell-based therapies,but safety concerns that complicate consideration for routine clinical use remain. Installing a safety switch" based on the inducible caspase-9 (iCASP9) suicide gene system should offer added control over undesirable cell replication or activity. Previous studies utilized lentiviral vectors to integrate the iCASP9 system into T cells and iPSCs. This method results in random genomic insertion of the suicide switch and inefficient killing of the cells after the switch is "turned on" with a small molecule (eg AP1903). To improve the safety and efficiency of the iCASP9 system for use in iPSC-based therapy we precisely installed the system into a genomic safe harbor the AAVS1 locus in the PPP1R12C gene. We then evaluated the efficiencies of different promoters to drive iCASP9 expression in human iPSCs. We report that the commonly used EF1$\alpha$ promoter is silenced in iPSCs and that the endogenous promoter of the PPP1R12C gene is not strong enough to drive high levels of iCASP9 expression. However the CAG promoter induces strong and stable iCASP9 expression in iPSCs and activation of this system with AP1903 leads to rapid killing and complete elimination of iPSCs and their derivatives including MSCs and chondrocytes in vitro. Furthermore iPSC-derived teratomas shrank dramatically or were completely eliminated after administration of AP1903 in mice. Our data suggest significant improvements on existing iCASP9 suicide switch technologies and may serve as a guide to other groups seeking to improve the safety of stem cell-based therapies." View Publication

This study traced intravenously administered induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (MSC) and assessed the impact of iPSC-MSC on preserving renal function in SD rat after 5/6 nephrectomy. The results of in vitro study showed that FeraTrack™Direct contrast particles (ie intracellular magnetic labelling) in the iPSC-MSC (ie iPS-MSCSPIONs ) were clearly identified by Prussian blue stain. Adult-male SD rats (n = 40) were categorized into group 1 (SC),group 2 [SC + iPS-MSCSPIONs (1.0 × 106 cells)/intravenous administration post-day-14 CKD procedure],group 3 (CKD),group 4 [CKD + iPS-MSCSPIONs (0.5 × 106 cells)] and group 5 [CKD + iPS-MSCSPIONs (1.0 × 106 cells)]. By day-15 after CKD induction,abdominal MRI demonstrated that iPS-MSCSPIONs were only in the CKD parenchyma of groups 4 and 5. By day 60,the creatinine level/ratio of urine protein to urine creatinine/kidney injury score (by haematoxylin and eosin stain)/fibrotic area (Masson's trichrome stain)/IF microscopic finding of kidney injury molecule-1 expression was lowest in groups 1 and 2,highest in group 3,and significantly higher in group 4 than in group 5,whereas IF microscopic findings of podocyte components (ZO-1/synaptopodin) and protein levels of anti-apoptosis ((Bad/Bcl-xL/Bcl-2) exhibited an opposite pattern to creatinine level among the five groups (all P {\textless} .0001). The protein expressions of cell-proliferation signals (PI3K/p-Akt/m-TOR,p-ERK1/2,FOXO1/GSK3$\beta$/p90RSK),apoptotic/DNA-damage (Bax/caspases8-10/cytosolic-mitochondria) and inflammatory (TNF-$\alpha$/TNFR1/TRAF2/NF-$\kappa$B) biomarkers displayed an identical pattern to creatinine level among the five groups (all P {\textless} .0001). The iPS-MSCSPIONs that were identified only in CKD parenchyma effectively protected the kidney against CKD injury. View Publication

PURPOSE Glioblastoma (GBM) is a fatal primary malignant brain tumor. GBM stem cells (GSC) contribute to resistance to the DNA-damaging chemotherapy,temozolomide. The epidermal growth factor receptor (EGFR) displays genomic alterations enabling DNA repair mechanisms in half of GBMs. We aimed to investigate EGFR/DNA combi-targeting in GBM. EXPERIMENTAL DESIGN ZR2002 is a combi-molecule" designed to inflict DNA damage through its chlorethyl moiety and induce irreversible EGFR tyrosine kinase inhibition. We assessed its in vitro efficacy in temozolomide-resistant patient-derived GSCs mesenchymal temozolomide-sensitive and resistant in vivo-derived GSC sublines and U87/EGFR isogenic cell lines stably expressing EGFR/wild-type or variant III (EGFRvIII). We evaluated its antitumor activity in mice harboring orthotopic EGFRvIII or mesenchymal TMZ-resistant GSC tumors. RESULTS ZR2002 induced submicromolar antiproliferative effects and inhibited neurosphere formation of all GSCs with marginal effects on normal human astrocytes. ZR2002 inhibited EGF-induced autophosphorylation of EGFR downstream Erk1/2 phosphorylation increased DNA strand breaks and induced activation of wild-type p53; the latter was required for its cytotoxicity through p53-dependent mechanism. ZR2002 induced similar effects on U87/EGFR cell lines and its oral administration significantly increased survival in an orthotopic EGFRvIII mouse model. ZR2002 improved survival of mice harboring intracranial mesenchymal temozolomide-resistant GSC line decreased EGFR Erk1/2 and AKT phosphorylation and was detected in tumor brain tissue by MALDI imaging mass spectrometry. CONCLUSIONS These findings provide the molecular basis of binary EGFR/DNA targeting and uncover the oral bioavailability blood-brain barrier permeability and antitumor activity of ZR2002 supporting potential evaluation of this first-in-class drug in recurrent GBM." View Publication

Emerging evidences have reported that periodontitis can be a risk factor for the pathogenesis of various systemic diseases. Porphyromonas gingivalis (Pg),one of the crucial pathogens in chronic periodontitis,has been spotlighted as a potential cause for the promotion and acceleration of periodontitis-associated systemic disorders. To investigate the association between Pg and intestinal disease or homeostasis,we treated Pg-derived lipopolysaccharide (LPS) in murine colitis model or intestinal organoid,respectively. Pg-derived LPS (Pg LPS) was administrated into chemically induced murine colitis model and disease symptoms were monitored compared with the infusion of LPS derived from E. coli (Ec LPS). Organoids isolated and cultured from mouse small intestine were treated with Pg or Ec LPS and further analyzed for the generation and composition of organoids. In vivo observations demonstrated that both Pg and Ec LPS exerted slight protective effects against murine colitis. Pg LPS did not affect the generation and growth of intestinal epithelial organoids. Among subtypes of epithelial cells,markers for stem cells,goblet cells or Paneth cells were changed in response to Pg LPS. Taken together,these results indicate that Pg LPS leads to partial improvement in colitis and that its treatment does not significantly affect the self-organization of intestinal organoids but may regulate the epithelial composition. View Publication