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BACKGROUND: The collagen gel contraction assay measures gel size to assess the contraction of cells embedded in collagen gel matrices. Using the assay with lung fibroblasts is useful in studying the lung tissue remodeling process in wound healing and disease development. However,the involvement of bronchial epithelial cells in this process should also be investigated. METHODS: We applied a layer of mucociliary differentiated bronchial epithelial cells onto collagen gel matrices with lung fibroblasts. This co-culture model enables direct contact between epithelial and mesenchymal cells. We stimulated the culture with transforming growth factor (TGF) beta1 as an inducer of tissue remodeling for 21 days,and measured gel size,histological changes,and expression of factors related to extracellular matrix homeostasis. RESULTS: TGF-beta1 exerted a concentration-dependent effect on collagen gel contraction and on contractile myofibroblasts in the mesenchymal collagen layer. TGF-beta1 also induced expression of the mesenchymal marker vimentin in the basal layer of the epithelium,suggesting the induction of epithelial-mesenchymal transition. In addition,the expression of various genes encoding extracellular matrix proteins was upregulated. Fibrotic tenascin-C accumulated in the sub-epithelial region of the co-culture model. CONCLUSION: Our findings indicate that TGF-beta1 can affect both epithelial and mesenchymal cells,and induce gel contraction and structural changes. Our novel in vitro co-culture model will be a useful tool for investigating the roles of epithelial cells,fibroblasts,and their interactions in the airway remodeling process. View Publication

Severe asthma represents a major unmet clinical need; understanding the pathophysiology is essential for the development of new therapies. Using microarray analysis,we previously found three immunological clusters in asthma: Th2-high,Th17-high,and Th2/17-low. Although new therapies are emerging for Th2-high disease,identifying molecular pathways in Th2-low disease remains an important goal. Further interrogation of our previously described microarray dataset revealed upregulation of gene expression for carcinoembryonic Ag cell adhesion molecule (CEACAM) family members in the bronchi of patients with severe asthma. Our aim was therefore to explore the distribution and cellular localization of CEACAM6 using immunohistochemistry on bronchial biopsy tissue obtained from patients with mild-to-severe asthma and healthy control subjects. Human bronchial epithelial cells were used to investigate cytokine and corticosteroid in vitro regulation of CEACAM6 gene expression. CEACAM6 protein expression in bronchial biopsies was increased in airway epithelial cells and lamina propria inflammatory cells in severe asthma compared with healthy control subjects. CEACAM6 in the lamina propria was localized to neutrophils predominantly. Neutrophil density in the bronchial mucosa was similar across health and the spectrum of asthma severity,but the percentage of neutrophils expressing CEACAM6 was significantly increased in severe asthma,suggesting the presence of an altered neutrophil phenotype. CEACAM6 gene expression in cultured epithelial cells was upregulated by wounding and neutrophil elastase. In summary,CEACAM6 expression is increased in severe asthma and primarily associated with airway epithelial cells and tissue neutrophils. CEACAM6 may contribute to the pathology of treatment-resistant asthma via neutrophil and airway epithelial cell-dependent pathways. View Publication

The respiratory tract with its ease of access,vast surface area and dense network of antigen-presenting cells (APCs) represents an ideal target for immune-modulation. Bio-mimetic nanocarriers such as virosomes may provide immunomodulatory properties to treat diseases such as allergic asthma. In our study we employed a triple co-culture model of epithelial cells,macrophages and dendritic cells to simulate the human airway barrier. The epithelial cell line 16HBE was grown on inserts and supplemented with human blood monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) for exposure to influenza virosomes and liposomes. Additionally,primary human nasal epithelial cells (PHNEC) and EpCAM+ epithelial progenitor cell mono-cultures were utilized to simulate epithelium from large and smaller airways,respectively. To assess particle uptake and phenotype change,cell cultures were analyzed by flow cytometry and pro-inflammatory cytokine concentrations were measured by ELISA. All cell types internalized virosomes more efficiently than liposomes in both mono- and co-cultures. APCs like MDMs and MDDCs showed the highest uptake capacity. Virosome and liposome treatment caused a moderate degree of activation in MDDCs from mono-cultures and induced an increased cytokine production in co-cultures. In epithelial cells,virosome uptake was increased compared to liposomes in both mono- and co-cultures with EpCAM+ epithelial progenitor cells showing highest uptake capacity. In conclusion,all cell types successfully internalized both nanocarriers with virosomes being taken up by a higher proportion of cells and at a higher rate inducing limited activation of MDDCs. Thus virosomes may represent ideal carrier antigen systems to modulate mucosal immune responses in the respiratory tract without causing excessive inflammatory changes. View Publication

INTRODUCTION: Lung cancer contains a small population of cancer stem cells that contribute to its initiation and progression. We investigated the biological function and clinical significance of aldehyde dehydrogenase 1A1 (ALDH1A1) in non-small-cell lung carcinoma (NSCLC). METHODS: ALDH1A1 assay or small interfering RNA transfection was employed to isolate ALDH1A1+ cells or knock down ALDH1A1 expression in H2087 cells,respectively. Biological functions of ALDH1A1+ and ALDH1A1 silenced cells were investigated using in vitro and in vivo methods. ALDH1A1 expression was analyzed using immunohistochemistry on tissue microarrays with 179 lung cancer tissues and 26 normal lung tissues. RESULTS: The abilities of clone formation,proliferation,cell growth,and migration were increased in ALDH1A1+ and ALDH1A1 silenced cells. ALDH1A1+ lung cancer cells initiated tumors that resembled the histopathologic characteristics and heterogeneity of the parental lung cancer cells in mice. The silencing of ALDH1A1 expression in H2087 lung cancer cells inhibited cell proliferation and migration significantly. ALDH1A1 was expressed in 42% of normal lung tissues (11 of 26),with strong expression in the basal cells and globular cells of the normal bronchus and weak expression in the alveolar epithelial cells. Compared with normal lung tissues,45% of NSCLC samples (81 of 179) were read as positive for ALDH1A1. Positive ALDH1A1 expression was correlated with patients' smoking status (p = 0.022),lymph-node metastasis (p = 0.006),clinical stage (p = 0.004),and a decreased overall survival time (p textless 0.001). Positive ALDH1A1 expression in lung cancer tissues was an independent prognostic factor for NSCLC (odds ratio = 5.232,p textless 0.001). CONCLUSION: Elucidating the biological functions of ALDH1A1 could be helpful in studying lung tumorigenesis and for developing new therapeutic approaches. View Publication

Respiratory syncytial virus (RSV) causes severe respiratory disease in young children. Antibodies specific for the RSV prefusion F protein have guided RSV vaccine research,and in human serum,these antibodies contribute to<90% of the neutralization response; however,detailed insight into the composition of the human B cell repertoire against RSV is still largely unknown. In order to study the B cell repertoire of three healthy donors for specificity against RSV,CD27+memory B cells were isolated and immortalized using BCL6 and Bcl-xL. Of the circulating memory B cells,0.35% recognized RSV-A2-infected cells,of which 59% were IgA-expressing cells and 41% were IgG-expressing cells. When we generated monoclonal B cells selected for high binding to RSV-infected cells,44.5% of IgG-expressing B cells and 56% of IgA-expressing B cells reacted to the F protein,while,unexpectedly,41.5% of IgG-expressing B cells and 44% of IgA expressing B cells reacted to the G protein. Analysis of the G-specific antibodies revealed that 4 different domains on the G protein were recognized. These epitopes predicted cross-reactivity between RSV strain A (RSV-A) and RSV-B and matched the potency of antibodies to neutralize RSV in HEp-2 cells and in primary epithelial cell cultures. G-specific antibodies were also able to induce antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis of RSV-A2-infected cells. However,these processes did not seem to depend on a specific epitope. In conclusion,healthy adults harbor a diverse repertoire of RSV glycoprotein-specific antibodies with a broad range of effector functions that likely play an important role in antiviral immunity.IMPORTANCEHuman RSV remains the most common cause of severe lower respiratory tract disease in premature babies,young infants,the elderly,and immunocompromised patients and plays an important role in asthma exacerbations. In developing countries,RSV lower respiratory tract disease has a high mortality. Without an effective vaccine,only passive immunization with palivizumab is approved for prophylactic treatment. However,highly potent RSV-specific monoclonal antibodies could potentially serve as a therapeutic treatment and contribute to disease control and mortality reduction. In addition,these antibodies could guide further vaccine development. In this study,we isolated and characterized several novel antibodies directed at the RSV G protein. This information can add to our understanding and treatment of RSV disease. View Publication

BACKGROUND In vitro systems of primary cystic fibrosis (CF) airway epithelial cells are an important tool to study molecular and functional features of the native respiratory epithelium. However,undifferentiated CF airway cell cultures grown under submerged conditions do not appropriately represent the physiological situation. A more advanced CF cell culture system based on airway epithelial cells grown at the air-liquid interface (ALI) recapitulates most of the in vivo-like properties but requires the use of invasive sampling methods. In this study,we describe a detailed characterization of fully differentiated primary CF airway epithelial cells obtained by non-invasive nasal brushing of pediatric patients. METHODS Differentiated cell cultures were evaluated with immunolabelling of markers for ciliated,mucus-secreting and basal cells,and tight junction and CFTR proteins. Epithelial morphology and ultrastructure was examined by histology and transmission electron microscopy. Ciliary beat frequency was investigated by a video-microscopy approach and trans-epithelial electrical resistance was assessed with an epithelial Volt-Ohm meter system. Finally,epithelial permeability was analysed by using a cell layer integrity test and baseline cytokine levels where measured by an enzyme-linked immunosorbent assay. RESULTS Pediatric CF nasal cultures grown at the ALI showed a differentiation into a pseudostratified epithelium with a mucociliary phenotype. Also,immunofluorescence analysis revealed the presence of ciliated,mucus-secreting and basal cells and tight junctions. CFTR protein expression was observed in CF (F508del/F508del) and healthy cultures and baseline interleukin (IL)-8 and IL-6 release were similar in control and CF ALI cultures. The ciliary beat frequency was 9.67 Hz and the differentiated pediatric CF epithelium was found to be functionally tight. CONCLUSION In summary,primary pediatric CF nasal epithelial cell cultures grown at the ALI showed full differentiation into ciliated,mucus-producing and basal cells,which adequately reflect the in vivo properties of the human respiratory epithelium. 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

Cellular plasticity contributes to the regenerative capacity of plants,invertebrates,teleost fishes and amphibians. In vertebrates,differentiated cells are known to revert into replicating progenitors,but these cells do not persist as stable stem cells. Here we present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. After the ablation of airway stem cells,we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts in repairing epithelial injury. Single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. By contrast,direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming,the propensity of committed cells to dedifferentiate is inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may have a more general role in the regeneration of many tissues and in multiple disease states,notably cancer. 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

Effective derivation of functional airway organoids from induced pluripotent stem cells (iPSCs) would provide valuable models of lung disease and facilitate precision therapies for airway disorders such as cystic fibrosis. However,limited understanding of human airway patterning has made this goal challenging. Here,we show that cyclical modulation of the canonical Wnt signaling pathway enables rapid directed differentiation of human iPSCs via an NKX2-1+progenitor intermediate into functional proximal airway organoids. We find that human NKX2-1+progenitors have high levels of Wnt activation but respond intrinsically to decreases in Wnt signaling by rapidly patterning into proximal airway lineages at the expense of distal fates. Using this directed approach,we were able to generate cystic fibrosis patient-specific iPSC-derived airway organoids with a defect in forskolin-induced swelling that is rescued by gene editing to correct the disease mutation. Our approach has many potential applications in modeling and drug screening for airway diseases. 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

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