技术资料

Langerhans cell histiocytosis (LCH) is a rare disease with an unknown etiology characterized by heterogeneous lesions containing CD207+CD1a+ cells that can arise in almost any tissue and cause significant morbidity and mortality. Precursors of pathological Langerhans cells have yet to be defined. Our aim was to identify circulating CD207+CD1a+ cells and their inducers in LCH. Expression of CD207 and CD1a in the blood myeloid compartment as well as thymic stromal lymphopoietin (TSLP) and transforming growth factor β (TGF-β) plasma levels were measured in 22 pediatric patients with active disease (AD) or nonactive disease (NAD). In patients with AD vs those with NAD,the myeloid compartment showed an increased CD11b (CD11bhigh plus CD11b+) fraction (39.7 ± 3.6 vs 18.6 ± 1.9),a higher percentage of circulating CD11bhighCD11c+CD207+ cells (44.5 ± 11.3 vs 3.2 ± 0.5),and the presence of CD11chighCD207+CD1a+ cells (25.0 ± 9.1 vs 2.3 ± 0.5). Blood CD207+CD1a+ cells were not observed in adult controls or umbilical cord. Increased TSLP and TGF-β levels were detected in patients with AD. Interestingly,plasma from patients with AD induces CD207 expression on CD14+ monocytes. We conclude that CD207+CD1a+ cells are circulating in patients with active LCH,and TSLP and TGF-β are potential drivers of Langerhans-like cells in vivo. View Publication

Mucosal Langerhans cells (LCs) originate from pre-dendritic cells and monocytes. However,the mechanisms involved in their in situ development remain unclear. Here,we demonstrate that the differentiation of murine mucosal LCs is a two-step process. In the lamina propria,signaling via BMP7-ALK3 promotes translocation of LC precursors to the epithelium. Within the epithelium,TGF-β1 finalizes LC differentiation,and ALK5 is crucial to this process. Moreover,the local microbiota has a major impact on the development of mucosal LCs,whereas LCs in turn maintain mucosal homeostasis and prevent tissue destruction. These results reveal the differential and sequential role of TGF-β1 and BMP7 in LC differentiation and highlight the intimate interplay of LCs with the microbiota. View Publication

Exposure to cigarette smoke causes a multitude of pathological changes leading to tissue damage and disease. Quantifying such changes in highly differentiated in vitro human tissue models may assist in evaluating the toxicity of tobacco products. In this methods development study,well-differentiated human air-liquid-interface (ALI) in vitro airway tissue models were used to assess toxicological endpoints relevant to tobacco smoke exposure. Whole mainstream smoke solutions (WSSs) were prepared from 2 commercial cigarettes (R60 and S60) that differ in smoke constituents when machine-smoked under International Organization for Standardization conditions. The airway tissue models were exposed apically to WSSs 4-h per day for 1-5 days. Cytotoxicity,tissue barrier integrity,oxidative stress,mucin secretion,and matrix metalloproteinase (MMP) excretion were measured. The treatments were not cytotoxic and had marginal effects on tissue barrier properties; however,other endpoints responded in time- and dose-dependent manners,with the R60 resulting in higher levels of response than the S60 for many endpoints. Based on the lowest effect dose,differences in response to the WSSs were observed for mucin induction and MMP secretion. Mitigation of mucin induction by cotreatment of cultures with N-acetylcysteine suggests that oxidative stress contributes to mucus hypersecretion. Overall,these preliminary results suggest that quantifying disease-relevant endpoints using ALI airway models is a potential tool for tobacco product toxicity evaluation. Additional research using tobacco samples generated under smoking machine conditions that more closely approximate human smoking patterns will inform further methods development. View Publication

A major challenge in developing gene-based therapies for airway diseases such as cystic fibrosis (CF) is sustaining therapeutic levels of transgene expression over time. This is largely due to airway epithelial cell turnover and the host immunogenicity to gene delivery vectors. Modern gene editing tools and delivery vehicles hold great potential for overcoming this challenge. There is currently not much known about how to deliver genes into airway stem cells,of which basal cells are the major type in human airways. In this study,helper-dependent adenoviral (HD-Ad) vectors were delivered to mouse and pig airways via intranasal delivery,and direct bronchoscopic instillation,respectively. Vector transduction was assessed by immunostaining of lung tissue sections,which revealed that airway basal cells of mice and pigs can be targeted in vivo. In addition,efficient transduction of primary human airway basal cells was verified with an HD-Ad vector expressing green fluorescent protein. Furthermore,we successfully delivered the human CFTR gene to airway basal cells from CF patients,and demonstrated restoration of CFTR channel activity following cell differentiation in air-liquid interface culture. Our results provide a strong rationale for utilizing HD-Ad vectors to target airway basal cells for permanent gene correction of genetic airway diseases. View Publication

A current model posits that cofilin-dependent actin severing negatively impacts dendritic spine volume. Studies suggested that increased cofilin activity underlies activity-dependent spine shrinkage,and that reduced cofilin activity induces activity-dependent spine growth. We suggest instead that both types of structural plasticity correlate with decreased cofilin activity. However,the mechanism of inhibition determines the outcome for spine morphology. RNAi in rat hippocampal cultures demonstrates that cofilin is essential for normal spine maintenance. Cofilin-F-actin binding and filament barbed-end production decrease during the early phase of activity-dependent spine shrinkage; cofilin concentration also decreases. Inhibition of the cathepsin B/L family of proteases prevents both cofilin loss and spine shrinkage. Conversely,during activity-dependent spine growth,LIM kinase stimulates cofilin phosphorylation,which activates phospholipase D-1 to promote actin polymerization. These results implicate novel molecular mechanisms and prompt a revision of the current model for how cofilin functions in activity-dependent structural plasticity. View Publication

Increasing evidence suggests that cellular stress may underlie mood disorders such as bipolar disorder and major depression,particularly as lithium and its targets can protect against neuronal cell death. Here we describe N-methyl-D-aspartate (NMDA)-induced filamentous actin reorganization (NIFAR) as a useful in-vitro model for studying acute neurocellular stress and investigating the effects of mood stabilizers. Brief incubation of cultured neurons with NMDA (50 µM,5 min) induces marked reorganization of F-actin within the somatodendritic domain of a majority of neurons. During NIFAR,F-actin is rapidly depleted from dendritic spines and aberrantly aggregates within the dendrite shaft. The widely used mood stabilizer lithium chloride prevented NIFAR in a time-dependent and dose-dependent manner,consistent with its known efficacy in treating bipolar disorder. Inhibitors of the lithium target glycogen synthase kinase 3 and its upstream activator phosphoinositide-3-kinase also prevented NIFAR. The antidepressant compounds imipramine and fluoxetine also attenuated NIFAR. These findings have potential relevance to neuropsychiatric diseases characterized by excessive glutamate receptor activity and synaptotoxicity. We propose that protection of the dendritic actin cytoskeleton may be a common mechanism shared by various mood stabilizers. View Publication

The discovery of induced pluripotent stem cells (iPSCs) and their application to patient-specific disease models offers new opportunities for studying the pathophysiology of neurological disorders. However,current methods for culturing iPSC-derived neuronal cells result in clustering of neurons,which precludes the analysis of individual neurons and defined neuronal networks. To address this challenge,cultures of human neurons on micropatterned surfaces are developed that promote neuronal survival over extended periods of time. This approach facilitates studies of neuronal development,cellular trafficking,and related mechanisms that require assessment of individual neurons and specific network connections. Importantly,micropatterns support the long-term stability of cultured neurons,which enables time-dependent analysis of cellular processes in living neurons. The approach described in this paper allows mechanistic studies of human neurons,both in terms of normal neuronal development and function,as well as time-dependent pathological processes,and provides a platform for testing of new therapeutics in neuropsychiatric disorders. View Publication

The myelination of axons is a crucial step during vertebrate central nervous system (CNS) development,allowing for rapid and energy efficient saltatory conduction of nerve impulses. Accordingly,the differentiation of oligodendrocytes,the myelinating cells of the CNS,and their expression of myelin genes are under tight transcriptional control. We previously identified a putative transcription factor,Myelin Regulatory Factor (Myrf),as being vital for CNS myelination. Myrf is required for the generation of CNS myelination during development and also for its maintenance in the adult. It has been controversial,however,whether Myrf directly regulates transcription,with reports of a transmembrane domain and lack of nuclear localization. Here we show that Myrf is a membrane-associated transcription factor that undergoes an activating proteolytic cleavage to separate its transmembrane domain-containing C-terminal region from a nuclear-targeted N-terminal region. Unexpectedly,this cleavage event occurs via a protein domain related to the autoproteolytic intramolecular chaperone domain of the bacteriophage tail spike proteins,the first time this domain has been found to play a role in eukaryotic proteins. Using ChIP-Seq we show that the N-terminal cleavage product directly binds the enhancer regions of oligodendrocyte-specific and myelin genes. This binding occurs via a defined DNA-binding consensus sequence and strongly promotes the expression of target genes. These findings identify Myrf as a novel example of a membrane-associated transcription factor and provide a direct molecular mechanism for its regulation of oligodendrocyte differentiation and CNS myelination. View Publication

Tissue-resident macrophages,such as microglia,Kupffer cells,and Langerhans cells,derive from Myb-independent yolk sac (YS) progenitors generated before the emergence of hematopoietic stem cells (HSCs). Myb-independent YS-derived resident macrophages self-renew locally,independently of circulating monocytes and HSCs. In contrast,adult blood monocytes,as well as infiltrating,gut,and dermal macrophages,derive from Myb-dependent HSCs. These findings are derived from the mouse,using gene knockouts and lineage tracing,but their applicability to human development has not been formally demonstrated. Here,we use human induced pluripotent stem cells (iPSCs) as a tool to model human hematopoietic development. By using a CRISPR-Cas9 knockout strategy,we show that human iPSC-derived monocytes/macrophages develop in an MYB-independent,RUNX1-,and SPI1 (PU.1)-dependent fashion. This result makes human iPSC-derived macrophages developmentally related to and a good model for MYB-independent tissue-resident macrophages,such as alveolar and kidney macrophages,microglia,Kupffer cells,and Langerhans cells. View Publication

Smoking is an important risk factor for the development of chronic obstructive pulmonary disease (COPD) and viral infections are believed to be major triggers of exacerbations,which periodically lead to a worsening of symptoms. The pro-inflammatory IL-1 family members IL-1α and IL-1β are increased in COPD patients and might contribute to disease pathology. We investigated whether individual or combined inhibition of these cytokines reduced lung inflammation in cigarette smoke (CS)-exposed and H1N1-infected BALB/c mice. Animals were treated with individual or combined antibodies (Abs) directed against IL-1α,IL-1β or IL-1R1. Cells in BAL fluid and cytokines/chemokines in lung homogenate were determined. The viral load was investigated. Blocking IL-1α had significant suppressive effects on total cells,neutrophils,and macrophages. Furthermore,it reduced KC levels significantly. Blocking of IL-1β did not provide significant activity. In primary human bronchial epithelial air-liquid-interface cell cultures infected with H1N1,IL-1α Abs but not IL-1β Abs reduced levels of TNF-α and IL-6. Concomitant usage of Abs against IL-1α/IL-1β revealed strong effects in vivo and reduced total cells,neutrophils and macrophages. Additionally,levels of KC,IL-6,TNF-α,MCP-1,MIP-1α and MIP-1β were significantly reduced and ICAM-1 and MUC5 A/C mRNA expression was attenuated. The viral load decreased significantly upon combined IL-1α/IL-1β Ab treatment. Blocking the IL-1R1 provided significant effects on total cells,neutrophils and macrophages but was inferior compared to inhibiting both its soluble ligands IL-1α/IL-1β. Our results suggest that combined inhibition of IL-1α/IL-1β might be beneficial to reduce CS/H1N1-induced airway inflammation. Moreover,combined targeting of both IL-1α/IL-1β might be more efficient compared to individual neutralization IL-1α or IL-1β or inhibition of the IL-1R1. View Publication

Synaptic cadherin adhesion complexes are known to be key regulators of synapse plasticity. However,the molecular mechanisms that coordinate activity-induced modifications in cadherin localization and adhesion and the subsequent changes in synapse morphology and efficacy remain unknown. We demonstrate that the intracellular cadherin binding protein δ-catenin is transiently palmitoylated by DHHC5 after enhanced synaptic activity and that palmitoylation increases δ-catenin-cadherin interactions at synapses. Both the palmitoylation of δ-catenin and its binding to cadherin are required for activity-induced stabilization of N-cadherin at synapses and the enlargement of postsynaptic spines,as well as the insertion of GluA1 and GluA2 subunits into the synaptic membrane and the concomitant increase in miniature excitatory postsynaptic current amplitude. Notably,context-dependent fear conditioning in mice resulted in increased δ-catenin palmitoylation,as well as increased δ-catenin-cadherin associations at hippocampal synapses. Together these findings suggest a role for palmitoylated δ-catenin in coordinating activity-dependent changes in synaptic adhesion molecules,synapse structure and receptor localization that are involved in memory formation. View Publication

The prion protein (PrP) is highly conserved and ubiquitously expressed,suggesting that it plays an important physiological function. However,despite decades of investigation,this role remains elusive. Here,by using animal and cellular models,we unveil a key role of PrP in the DNA damage response. Exposure of neurons to a genotoxic stress activates PRNP transcription leading to an increased amount of PrP in the nucleus where it interacts with APE1,the major mammalian endonuclease essential for base excision repair,and stimulates its activity. Preventing the induction of PRNP results in accumulation of abasic sites in DNA and impairs cell survival after genotoxic treatment. Brains from Prnp(-/-) mice display a reduced APE1 activity and a defect in the repair of induced DNA damage in vivo. Thus,PrP is required to maintain genomic stability in response to genotoxic stresses. View Publication