技术资料

Asthma is highly prevalent,but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study,we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore,deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17,miR-144,and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment,while it's expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally,we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies. View Publication

We inhale respiratory pathogens continuously,and the subsequent signaling events between host and microbe are complex,ultimately resulting in clearance of the microbe,stable colonization of the host,or active disease. Traditional in vitro methods are ill-equipped to study these critical events in the context of the lung microenvironment. Here we introduce a microscale organotypic model of the human bronchiole for studying pulmonary infection. By leveraging microscale techniques,the model is designed to approximate the structure of the human bronchiole,containing airway,vascular,and extracellular matrix compartments. To complement direct infection of the organotypic bronchiole,we present a clickable extension that facilitates volatile compound communication between microbial populations and the host model. Using Aspergillus fumigatus,a respiratory pathogen,we characterize the inflammatory response of the organotypic bronchiole to infection. Finally,we demonstrate multikingdom,volatile-mediated communication between the organotypic bronchiole and cultures of Aspergillus fumigatus and Pseudomonas aeruginosa. View Publication

Extracellular matrix (ECM) composition and stiffness are major driving forces for the development and persistence of fibrotic diseases. Lysyl oxidase (LOX) and LOX-like (LOXL) proteins play crucial roles in ECM remodeling due to their collagen crosslinking and intracellular functions. Here,we systematically investigated LOX/L expression in primary fibroblasts and epithelial cells under fibrotic conditions,Bleomycin (BLM) induced lung fibrosis and in human IPF tissue. Basal expression of all LOX/L family members was detected in epithelial cells and at higher levels in fibroblasts. Various pro-fibrotic stimuli broadly induced LOX/L expression in fibroblasts,whereas specific induction of LOXL2 and partially LOX was observed in epithelial cells. Immunohistochemical analysis of lung tissue from 14 IPF patients and healthy donors revealed strong induction of LOX and LOXL2 in bronchial and alveolar epithelium as well as fibroblastic foci. Using siRNA experiments we observed that LOXL2 and LOXL3 were crucial for fibroblast-to-myofibroblast transition (FMT). As FMT could only be reconstituted with an enzymatically active LOXL2 variant,we conclude that LOXL2 enzymatic function is crucial for fibroblast transdifferentiation. In summary,our study provides a comprehensive analysis of the LOX/L family in fibrotic lung disease and indicates prominent roles for LOXL2/3 in fibroblast activation and LOX/LOXL2 in IPF. View Publication

3D constructs composed of differentiated immortalized primary normal human bronchial epithelial (NHBE) cells (CL-1548) were repeatedly exposed at the air-liquid interface to non-lethal concentrations of mainstream cigarette smoke (4 cigarettes a day,5days/week,8 repetitions in total) and e-cigarette vapor (50 puffs a day,5 days/week,8 repetitions in total) to build up a permanent burden on the cells. Samples were taken after 4,6 and 8 times of repeated smoke exposure and the cultures were investigated using histopathological methods Compared to the clean air-exposed cultures (process control) and incubator control,the aerosol-exposed cultures showed a reduction of ciliated,mucus-producing and club cells. At the end of the exposure phase,we even found metaplastic areas positive for CK13 antibody in the cultures exposed to mainstream cigarette smoke and e-liquid vapor,commonly seen in squamous cells as a marker for non-cornified squamous epithelium. The control cultures (incubator cells) showed no comparable phenotypical changes. In conclusion,our in vitro model presents a valuable tool to study the induction of phenotypical changes after exposure to hazardous airborne material. View Publication

To better characterize biological responses to atmospheric organic aerosols,the efficient delivery of aerosol to in vitro lung cells is necessary. In this study,chamber generated secondary organic aerosol (SOA) entered the commercialized exposure chamber (CULTEX® Radial Flow System Compact) where it interfaced with an electrostatic precipitator (ESP) (CULTEX® Electrical Deposition Device) and then deposited on a particle collection plate. This plate contained human lung cells (BEAS-2B) that were cultured on a membrane insert to produce an air-liquid interface (ALI). To augment in vitro assessment using the ESP exposure device,the particle dose was predicted for various sampling parameters such as particle size,ESP deposition voltage,and sampling flowrate. The dose model was evaluated against the experimental measured mass of collected airborne particles. The high flowrate used in this study increased aerosol dose but failed to achieve cell stability. For example,RNA in the ALI BEAS-2B cells in vitro was stable at 0.15 L/minute but decayed at high flowrates. The ESP device and the resulting model were applied to in vitro studies (i.e.,viability and IL-8 expression) of toluene SOA using ALI BEAS-2B cells with a flowrate of 0.15 L/minute,and no cellular RNA decay occurred. View Publication

Eicosanoids are a group of bioactive lipids that are shown to be important mediators of neutrophilic inflammation; selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases,including cystic fibrosis,are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 is a pathogen-elicited epithelial-produced neutrophil chemoattractant that directs transepithelial migration in response to infection. Following hepoxilin A3-driven transepithelial migration,neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid,leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2,and the cytosolic phospholipase A2 (cPLA2)α isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of Pseudomonas aeruginosa-induced neutrophil transepithelial migration has not been explored. Human and mouse neutrophil-epithelial cocultures were used to evaluate the role of neutrophil-derived cPLA2α in infection-induced transepithelial signaling by pharmacological and genetic approaches. Primary human airway basal stem cell-derived epithelial cultures and micro-optical coherence tomography,a new imaging modality that captures two- and three-dimensional real-time dynamics of neutrophil transepithelial migration,were applied. Evidence from these studies suggests that cPLA2α expressed by neutrophils,but not epithelial cells,plays a significant role in infection-induced neutrophil transepithelial migration by mediating LTB4 synthesis during migration,which serves to amplify the magnitude of neutrophil recruitment in response to epithelial infection. View Publication

Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 μm pore-sized transwells,compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production,consistent with conventional ALIs,as visualized by micro-optical coherence tomography (μOCT). μOCT is a recently developed imaging modality with the capacity for real time two- A nd three-dimensional analysis of cellular events in marked detail,including neutrophil transmigratory dynamics. Further,the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients,and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with μOCT imaging offers significant opportunity to probe,in great detail,micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface. View Publication

Human bocavirus type-1 (HBoV1) has a high tropism for the apical membrane of human airway epithelia. The packaging of a recombinant adeno-associated virus 2 (rAAV2) genome into HBoV1 capsid produces a chimeric vector (rAAV2/HBoV1) that also efficiently transduces human airway epithelia. As such,this vector is attractive for use in gene therapies to treat lung diseases such as cystic fibrosis. However,preclinical development of rAAV2/HBoV1 vectors has been hindered by the fact that humans are the only known host for HBoV1 infection. This study reports that rAAV2/HBoV1 vector is capable of efficiently transducing the lungs of both newborn (3- to 7-day-old) and juvenile (29-day-old) ferrets,predominantly in the distal airways. Analyses of in vivo,ex vivo,and in vitro models of the ferret proximal airway demonstrate that infection of this particular region is less effective than it is in humans. Studies of vector binding and endocytosis in polarized ferret proximal airway epithelial cultures revealed that a lack of effective vector endocytosis is the main cause of inefficient transduction in vitro. While transgene expression declined proportionally with growth of the ferrets following infection at 7 days of age,reinfection of ferrets with rAAV2/HBoV1 at 29 days gave rise to approximately 5-fold higher levels of transduction than observed in naive infected 29-day-old animals. The findings presented here lay the foundation for clinical development of HBoV1 capsid-based vectors for lung gene therapy in cystic fibrosis using ferret models. View Publication

Human rhinovirus (HRV) is the most common virus contributing to acute exacerbations of chronic obstructive pulmonary disease (COPD) nearly year-round,but the mechanisms have not been well elucidated. Recent clinical studies suggest that high levels of growth differentiation factor 15 (GDF15) protein in the blood are associated with an increased yearly rate of all-cause COPD exacerbations. Therefore,in the current study,we investigated whether GDF15 promotes HRV infection and virus-induced lung inflammation. We first examined the role of GDF15 in regulating host defense and HRV-induced inflammation using human GDF15 transgenic mice and cultured human GDF15 transgenic mouse tracheal epithelial cells. Next,we determined the effect of GDF15 on viral replication,antiviral responses,and inflammation in human airway epithelial cells with GDF15 knockdown and HRV infection. Finally,we explored the signaling pathways involved in airway epithelial responses to HRV infection in the context of GDF15. Human GDF15 protein over-expression in mice led to exaggerated inflammatory responses to HRV,increased infectious particle release,and decreased IFN-λ2/3 (IL-28A/B) mRNA expression in the lung. Moreover,GDF15 facilitated HRV replication and inflammation via inhibiting IFN-λ1/IL-29 protein production in human airway epithelial cells. Lastly,Smad1 cooperated with interferon regulatory factor 7 (IRF7) to regulate airway epithelial responses to HRV infection partly via GDF15 signaling. Our results reveal a novel function of GDF15 in promoting lung HRV infection and virus-induced inflammation,which may be a new mechanism for the increased susceptibility and severity of respiratory viral (i.e.,HRV) infection in cigarette smoke-exposed airways with GDF15 over-production. View Publication

Organoids represent both a potentially powerful tool for the study cell-cell interactions within tissue-like environments,and a platform for tissue regenerative approaches. The development of lung tissue-like organoids from human adult-derived cells has not previously been reported. Here we combined human adult primary bronchial epithelial cells,lung fibroblasts,and lung microvascular endothelial cells in supportive 3D culture conditions to generate airway organoids. We demonstrate that randomly-seeded mixed cell populations undergo rapid condensation and self-organization into discrete epithelial and endothelial structures that are mechanically robust and stable during long term culture. After condensation airway organoids generate invasive multicellular tubular structures that recapitulate limited aspects of branching morphogenesis,and require actomyosin-mediated force generation and YAP/TAZ activation. Despite the proximal source of primary epithelium used in the airway organoids,discrete areas of both proximal and distal epithelial markers were observed over time in culture,demonstrating remarkable epithelial plasticity within the context of organoid cultures. Airway organoids also exhibited complex multicellular responses to a prototypical fibrogenic stimulus (TGF-??1) in culture,and limited capacity to undergo continued maturation and engraftment after ectopic implantation under the murine kidney capsule. These results demonstrate that the airway organoid system developed here represents a novel tool for the study of disease-relevant cell-cell interactions,and establishes this platform as a first step toward cell-based therapy for chronic lung diseases based on de novo engineering of implantable airway tissues. View Publication

OBJECTIVE To evaluate the effects of MUC18 on IL-13-mediated airway inflammatory responses in human airway epithelial cells and in mice. MATERIALS Primary normal human tracheobronchial epithelial (HTBE) cells,wild-type (WT) and Muc18 knockout (KO) mice,and mouse tracheal epithelial cells (mTECs) were utilized. TREATMENT Cultured HTBE cells treated with MUC18 siRNA or MUC18 expressing lentivirus were incubated with IL-13 (10 ng/mL) for 24 h. Mice were intranasally instilled with 500 ng of IL-13 for 3 days. mTECs were treated with IL-13 (10 ng/mL) for 3 days. METHODS PCR was used to measure mRNA expression. Western Blot and ELISAs were used to quantify protein expression. Cytospins of bronchoalveolar lavage (BAL) cells were used to obtain leukocyte differentials. RESULTS MUC18 siRNA reduced IL-13-mediated eotaxin-3 (183 ± 44 vs. 380 ± 59 pg/mL,p < 0.05),while MUC18 overexpression increased IL-13-mediated eotaxin-3 (95 ± 3 vs. 58 ± 3 pg/mL,p < 0.05) in HTBE cells. IL-13-treated Muc18 KO mice had a lower percentage of neutrophils in BAL than WT mice (25 ± 3 vs. 35 ± 3%,p = 0.0565). CONCLUSIONS These results implicate MUC18 as a potential enhancer of airway inflammation in a type 2 cytokine (e.g.,IL-13) milieu. View Publication

When inhaled,ozone (O3) interacts with cholesterols of airway epithelial cell membranes or the lung lining fluid,generating chemically reactive oxysterols. The mechanism by which O3-derived oxysterols affect molecular function is unknown. Our data show that in vitro exposure of human bronchial epithelial cells to O3 results in the formation of oxysterols,epoxycholesterol-α and β (α-EpCh,β-EpCh) and Secosterol A and B (Seco A,SecoB),in cell lysates and apical washes. Similarly,bronchoalveolar lavage fluid obtained from human volunteers exposed to O3 contained elevated levels of these oxysterol species. As expected,O3-derived oxysterols have a pro-inflammatory effect and increase NF-κB activity. Interestingly,expression of the cholesterol efflux pump ATP-binding cassette transporter 1 (ABCA1),which is regulated by activation of the liver X receptor (LXR),was suppressed in epithelial cells exposed to O3. Additionally,exposure of LXR knockout mice to O3 enhanced pro-inflammatory cytokine production in the lung,suggesting LXR inhibits O3-induced inflammation. Using alkynyl surrogates of O3-derived oxysterols,our data demonstrate adduction of LXR with Seco A. Similarly,supplementation of epithelial cells with alkynyl-tagged cholesterol followed by O3 exposure causes observable lipid-LXR adduct formation. Experiments using Seco A and the LXR agonist T0901317 (T09) showed reduced expression of ABCA1 as compared to stimulation with T09 alone,indicating that Seco A-LXR protein adduct formation inhibits LXR activation by traditional agonists. Overall,these data demonstrate that O3-derived oxysterols have pro-inflammatory functions and form lipid-protein adducts with LXR,thus leading to suppressed cholesterol regulatory gene expression and providing a biochemical mechanism mediating O3-derived formation of oxidized lipids in the airways and subsequent adverse health effects. View Publication