The extracellular matrix (ECM) is an important contributor to the asthmatic phenotype. Recent studies investigating airway inflammation have demonstrated an association between hyaluronan (HA) accumulation and inflammatory cell infiltration of the airways. The ECM proteoglycan versican interacts with HA and is important in the recruitment and activation of leukocytes during inflammation. We investigated the role of versican in the pathogenesis of asthmatic airway inflammation. Using cockroach antigen (CRA)-sensitized murine models of allergic asthma, we demonstrate increased subepithelial versican in the airways of CRA-treated mice that parallels subepithelial increases in HA and leukocyte infiltration. During the acute phase, CRA-treated mice displayed increased gene expression of the four major versican isoforms, as well as increased expression of HA synthases. Furthermore, in a murine model that examines both acute and chronic CRA exposure, versican staining peaked 8 days following CRA challenge and preceded subepithelial leukocyte infiltration. We also assessed versican and HA expression in differentiated primary human airway epithelial cells from asthmatic and healthy children. Increases in the expression of versican isoforms and HA synthases in these epithelial cells were similar to those of the murine model. These data indicate an important role for versican in the establishment of airway inflammation in asthma. View Publication

Mycoplasma pneumoniae is a human respiratory tract pathogen causing acute and chronic airway disease states that can include long-term carriage and extrapulmonary spread. The mechanisms of persistence and migration beyond the conducting airways, however, remain poorly understood. We previously described an acute exposure model using normal human bronchial epithelium (NHBE) in air-liquid interface culture, showing that M. pneumoniae gliding motility is essential for initial colonisation and subsequent spread, including localisation to epithelial cell junctions. We extended those observations here, characterizing M. pneumoniae infection of NHBE for up to 4 weeks. Colonisation of the apical surface was followed by pericellular invasion of the basolateral compartment and migration across the underlying transwell membrane. Despite fluctuations in transepithelial electrical resistance and increased NHBE cell desquamation, barrier function remained largely intact. Desquamation was accompanied by epithelial remodelling that included cytoskeletal reorganisation and development of deep furrows in the epithelium. Finally, M. pneumoniae strains S1 and M129 differed with respect to invasion and histopathology, consistent with contrasting virulence in experimentally infected mice. In summary, this study reports pericellular invasion, NHBE cytoskeletal reorganisation, and tissue remodelling with persistent infection in a human airway epithelium model, providing clear insight into the likely route for extrapulmonary spread. View Publication

The embryonic mouse lung is a widely used substitute for human lung development. For example, attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology, gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover, we identify mouse-human differences, including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated. View Publication

The combination therapy of lumacaftor and ivacaftor (Orkambi®) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508 It has been predicted that Orkambi® could treat patients with rarer mutations of similar theratype"; however a standardized approach confirming efficacy in these cohorts has not been reported. Here we demonstrate that patients bearing the rare mutation: c.3700 A>G causing protein misprocessing and altered channel function-similar to ΔF508-CFTR are unlikely to yield a robust Orkambi® response. While in silico and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor respectively this combination led to a minor in vitro response in patient-derived tissue. A CRISPR/Cas9-edited bronchial epithelial cell line bearing this mutation enabled studies showing that an "amplifier" compound effective in increasing the levels of immature CFTR protein augmented the Orkambi® response. Importantly this "amplifier" effect was recapitulated in patient-derived nasal cultures-providing the first evidence for its efficacy in augmenting Orkambi® in tissues harboring a rare CF-causing mutation. We propose that this multi-disciplinary approach including creation of CRISPR/Cas9-edited cells to profile modulators together with validation using primary tissue will facilitate therapy development for patients with rare CF mutations. 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

Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples, while requiring low cell numbers. To date, a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative, EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris, doublets and dead cells from the analysis. For validation, the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers, HBEC recovered from BAL (2.3% of live cells), BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed, validated, and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases. View Publication

In vitro models of human bronchial epithelium are useful for toxicological testing because of their resemblance to in vivo tissue. We constructed a model of human bronchial tissue which has a fibroblast layer embedded in a collagen matrix directly below a fully-differentiated epithelial cell layer. The model was applied to whole cigarette smoke (CS) exposure repeatedly from an air-liquid interface culture while bronchial epithelial cells were differentiating. The effects of CS exposure on differentiation were determined by histological and gene expression analyses on culture day 21. We found a decrease in ciliated cells and perturbation of goblet cell differentiation. We also analyzed the effects of CS exposure on the inflammatory response, and observed a significant increase in secretion of IL-8, GRO-α, IL-1β, and GM-CSF. Interestingly, secretion of these mediators was augmented with repetition of whole CS exposure. Our data demonstrate the usefulness of our bronchial tissue model for in vitro testing and the importance of exposure repetition in perturbing the differentiation and inflammation processes. View Publication

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

Pulmonary disease is the major cause of morbidity and mortality in patients with cystic fibrosis, a disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Heterogeneity in CFTR genotype-phenotype relationships in affected individuals plus the escalation of drug discovery targeting specific mutations highlights the need to develop robust in vitro platforms with which to stratify therapeutic options using relevant tissue. Toward this goal, we adapted a fluorescence plate reader assay of apical CFTR-mediated chloride conductance to enable profiling of a panel of modulators on primary nasal epithelial cultures derived from patients bearing different CFTR mutations. This platform faithfully recapitulated patient-specific responses previously observed in the gold-standard but relatively low-throughput Ussing chamber. Moreover using this approach we identified a novel strategy with which to augment the response to an approved drug in specific patients. In proof of concept studies we also validated the use of this platform in measuring drug responses in lung cultures differentiated from cystic fibrosis iPS cells. Taken together we show that this medium throughput assay of CFTR activity has the potential to stratify cystic fibrosis patient-specific responses to approved drugs and investigational compounds in vitro in primary and iPS cell-derived airway cultures. View Publication

Aberrant antioxidant activity and excessive deposition of extracellular matrix (ECM) are hallmarks of interstitial lung diseases (ILD). It is known that oxidative stress alters the ECM, but extracellular antioxidant defence mechanisms in ILD are incompletely understood. Here, we extracted abundance and detergent solubility of extracellular antioxidant enzymes from a proteomic dataset of bleomycin-induced lung fibrosis in mice and assessed regulation and distribution of glutathione peroxidase 3 (GPX3) in murine and human lung fibrosis. Superoxide dismutase 3 (Sod3), Gpx3, and Gpx activity were increased in mouse BALF during bleomycin-induced lung fibrosis. In lung tissue homogenates, Gpx3, but not Sod3, was upregulated and detergent solubility profiling indicated that Gpx3 associated with ECM proteins. Immunofluorescence analysis showed that Gpx3 was expressed by bronchial epithelial cells and interstitial fibroblasts and localized to the basement membrane and interstitial ECM in lung tissue. As to human ILD samples, BALF of some patients contained high levels of GPX3, and GPX3 was upregulated in lung homogenates from IPF patients. GPX3 expression in primary human bronchial epithelial cells and lung fibroblasts was downregulated by TNF-$$, but more variably regulated by TGF-$$1 and menadione. In conclusion, the antioxidant enzyme GPX3 localizes to lung ECM and is variably upregulated in ILD. View Publication

Graphical Abstract Highlights d SMAD activity is active in suprabasal cells but is weaker in basal epithelial cells d SMAD signaling activity correlates with mucociliary differentiation in the airway d Dual TGFb/BMP inhibition prevents spontaneous differentiation in culture d Dual TGFb/BMP inhibition allows prolonged culture of diverse epithelial basal cells Correspondence jrajagopal@partners.org In Brief Mou et al. show that small-molecule-mediated SMAD signaling inhibition allows prolonged feeder-free culture of diverse functional epithelial basal stem cells in a 2D format. This methodology provides a facile patient-specific epithelial disease modeling platform, as shown by the expansion of airway epithelium from non-invasively obtained specimens from cystic fibrosis patients. View Publication

We have established an in vitro 3D system which recapitulates the human tracheo-bronchial mucosa comprehensive of the pseudostratified epithelium and the underlying stromal tissue. In particular, we reported that the mature model, entirely constituted of primary cells of human origin, develops key markers proper of the native tissue such as the mucociliary differentiation of the epithelial sheet and the formation of the basement membrane. The infection of the pseudo-tissue with a strain of NonTypeable Haemophilus influenzae results in bacteria association and crossing of the mucus layer leading to an apparent targeting of the stromal space where they release large amounts of vesicles and form macro-structures. In summary, we propose our in vitro model as a reliable and potentially customizable system to study mid/long term host-pathogen processes. View Publication