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Development of effective treatment strategies for lung tissue destruction as seen in emphysema would greatly benefit from representative human in vitro models of the alveolar compartment. Studying how cellular cross talk and/or (altered) biomechanical cues affect alveolar epithelial function could provide new insight for tissue repair strategies. Preclinical models of the alveolus ideally combine human primary patient-derived lung cells with advanced cell culture applications such as breathing-related stretch, to reliably represent the alveolar microenvironment. To test the feasibility of such a model, we isolated primary alveolar type 2 cells (AEC2s) from patient-derived lung tissues including those from patients with severe emphysema, using magnetic bead-based selection of cells expressing the AEC2 marker HTII-280. We obtained pure alveolar feeder-free organoid cultures using a minimally modified commercial medium. This was confirmed by known AEC2 markers as well as by detection of lamellar bodies using electron microscopy. Following (organoid-based) expansion, cells were seeded on both cell culture inserts and the Chip-S1 Organ-Chip that has a flexible polydimethylsiloxane (PDMS) membrane enabling the application of dynamic stretch. AEC2s cultured for 7 days on inserts or the chip maintained expression of HTII-280, prosurfactant protein C (SP-C), SP-A and SP-B, and zonula occludens-1 (ZO-1) also in the presence of stretch. AEC2s cultured on the chip showed lower expression levels of epithelial-mesenchymal transition-related vimentin expression compared with static cultures on inserts. The combination of a straightforward culture method of patient-derived AEC2s and their application in microfluidic chip cultures supports successful development of more representative human preclinical models of the (diseased) alveolar compartment.

Air-liquid interface (ALI) cultures are frequently used in lung research but require substantial cell numbers that cannot readily be obtained from patients. We explored whether organoid expansion [three-dimensional (3D)] can be used to establish ALI cultures from clinical samples with low epithelial cell numbers. Airway epithelial cells were obtained from tracheal aspirates (TA) from preterm newborns and from bronchoalveolar lavage (BAL) or bronchial tissue (BT) from adults. TA and BAL cells were 3D-expanded, whereas cells from BT were expanded in 3D and 2D. Following expansion, cells were cultured at ALI to induce differentiation. The impact of cell origin and 2D or 3D expansion was assessed with respect to 1 ) cellular composition, 2 ) response to cigarette smoke exposure, and 3 ) effect of Notch inhibition or IL-13 stimulation on cellular differentiation. We established well-differentiated ALI cultures from all samples. Cellular compositions (basal, ciliated, and goblet cells) were comparable. All 3D-expanded cultures showed a similar stress response following cigarette smoke exposure but differed from the 2D-expanded cultures. Higher peak levels of antioxidant genes HMOX1 and NQO1 and a more rapid return to baseline, and a lower unfolded protein response was observed after cigarette smoke exposure in 3D-derived cultures compared to 2D-derived cultures. In addition, TA- and BAL-derived cultures were less sensitive to modulation by DAPT or IL-13 than BT-derived cultures. Organoid-based expansion of clinical samples with low cell numbers, such as TA from preterm newborns is a valid method and tool to establish ALI cultures.

Disturbances in mitochondrial structure and function in lung epithelial cells have been implicated in the pathogenesis of various lung diseases, including chronic obstructive pulmonary disease (COPD). Such disturbances affect not only cellular energy metabolism but also alter a range of indispensable cellular homeostatic functions in which mitochondria are known to be involved. These range from cellular differentiation, cell death pathways, and cellular remodeling to physical barrier function and innate immunity, all of which are known to be impacted by exposure to cigarette smoke and have been linked to COPD pathogenesis. Next to their well-established role as the first physical frontline against external insults, lung epithelial cells are immunologically active. Malfunctioning epithelial cells with defective mitochondria are unable to maintain homeostasis and respond adequately to further stress or injury, which may ultimately shape the phenotype of lung diseases. In this review, we provide a comprehensive overview of the impact of cigarette smoke on the development of mitochondrial dysfunction in the lung epithelium and highlight the consequences for cell function, innate immune responses, epithelial remodeling, and epithelial barrier function in COPD. We also discuss the applicability and potential therapeutic value of recently proposed strategies for the restoration of mitochondrial function in the treatment of COPD.

Transforming growth factor-β (TGF-β)-induced fibroblast-to-myofibroblast differentiation contributes to remodeling in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis, but whether this impacts the ability of fibroblasts to support lung epithelial repair remains little explored. We pretreated human lung fibroblasts [primary (phFB) or MRC5 cells] with recombinant human TGF-β to induce myofibroblast differentiation, then cocultured them with adult mouse lung epithelial cell adhesion molecule-positive cells (EpCAM + ) to investigate their capacity to support epithelial organoid formation in vitro. While control phFB and MRC5 lung fibroblasts supported organoid formation of mouse EpCAM + cells, TGF-β pretreatment of both phFB and MRC5 impaired organoid-supporting ability. We performed RNA sequencing of TGF-β-treated phFB, which revealed altered expression of key Wnt signaling pathway components and Wnt/β-catenin target genes, and modulated expression of secreted factors involved in mesenchymal-epithelial signaling. TGF-β profoundly skewed the transcriptional program induced by the Wnt/β-catenin activator CHIR99021. Supplementing organoid culture media recombinant hepatocyte growth factor or fibroblast growth factor 7 promoted organoid formation when using TGF-β pretreated fibroblasts. In conclusion, TGF-β-induced myofibroblast differentiation results in Wnt/β-catenin pathway skewing and impairs fibroblast ability to support epithelial repair likely through multiple mechanisms, including modulation of secreted growth factors.

Diesel emissions are the main source of air pollution in urban areas, and diesel exposure is linked with substantial adverse health effects. In vitro diesel exposure models are considered a suitable tool for understanding these effects. Here we aimed to use a controlled in vitro exposure system to whole diesel exhaust to study the effect of whole diesel exhaust concentration and exposure duration on mucociliary differentiated human primary bronchial epithelial cells (PBEC). PBEC cultured at the air-liquid interface were exposed for 60 to 375 min to three different dilutions of diesel exhaust (DE). The DE mixture was generated by an engine at 47% load, and characterized for particulate matter size and distribution and chemical and gas composition. Cytotoxicity and epithelial barrier function was assessed, as well as mRNA expression and protein release analysis. DE caused a significant dose-dependent increase in expression of oxidative stress markers (HMOX1 and NQO1; n = 4) at 6 h after 150 min exposure. Furthermore, DE significantly increased the expression of the markers of the integrated stress response CHOP and GADD34 and of the proinflammatory chemokine CXCL8, as well as release of CXCL8 protein. Cytotoxic effects or effects on epithelial barrier function were observed only after prolonged exposures to the highest DE dose. These results demonstrate the suitability of our model and that exposure dose and duration and time of analysis postexposure are main determinants for the effects of DE on differentiated primary human airway epithelial cells., (Copyright © 2016 the American Physiological Society.)

Patients with chronic obstructive pulmonary disease (COPD) have an increased risk for cardiovascular disease (CVD). Currently, COPD patients with atherosclerosis (i.e., the most important underlying cause of CVD) receive COPD therapy complemented with standard CVD therapy. This may, however, not be the most optimal treatment. To investigate the link between COPD and atherosclerosis and to develop specific therapeutic strategies for COPD patients with atherosclerosis, a substantial number of preclinical studies using murine models have been performed. In this review, we summarize the currently used murine models of COPD and atherosclerosis, both individually and combined, and discuss the relevance of these models for studying the pathogenesis and development of new treatments for COPD patients with atherosclerosis. Murine and clinical studies have provided complementary information showing a prominent role for systemic inflammation and oxidative stress in the link between COPD and atherosclerosis. These and other studies showed that murine models for COPD and atherosclerosis are useful tools and can provide important insights relevant to understanding the link between COPD and CVD. More importantly, murine studies provide good platforms for studying the potential of promising (new) therapeutic strategies for COPD patients with CVD., (Copyright © 2016 the American Physiological Society.)

Supplemental oxygen after premature birth results in aberrant airway, alveolar, and pulmonary vascular development with an increased risk for bronchopulmonary dysplasia, and development of wheeze and asthma, pulmonary hypertension, and chronic obstructive pulmonary disease in survivors. Although stimulation of the nitric oxide (NO)-soluble guanylate cyclase (sGC)-cGMP signal transduction pathway has significant beneficial effects on disease development in animal models, so far this could not be translated to the clinic. Oxidative stress reduces the NO-sGC-cGMP pathway by oxidizing heme-bound sGC, resulting in inactivation or degradation of sGC. Reduced sGC activity and/or expression is associated with pathology due to premature birth, oxidative stress-induced lung injury, including impaired alveolar maturation, smooth muscle cell (SMC) proliferation and contraction, impaired airway relaxation and vasodilation, inflammation, pulmonary hypertension, right ventricular hypertrophy, and an aggravated response toward hyperoxia-induced neonatal lung injury. Recently, Britt et al. (10) demonstrated that histamine-induced Ca(2+) responses were significantly elevated in hyperoxia-exposed fetal human airway SMCs compared with normoxic controls and that this hyperoxia-induced increase in the response was strongly reduced by NO-independent stimulation and activation of sGC. These recent studies highlight the therapeutic potential of sGC modulators in the treatment of preterm infants for respiratory distress with supplemental oxygen. Such treatment is aimed at improving aberrant alveolar and vascular development of the neonatal lung and preventing the development of wheezing and asthma in survivors of premature birth. In addition, these studies highlight the suitability of fetal human airway SMCs as a translational model for pathological airway changes in the neonate., (Copyright © 2015 the American Physiological Society.)

Anticholinergics, blocking the muscarinic M3 receptor, are effective bronchodilators for patients with chronic obstructive pulmonary disease. Recent evidence from M(3) receptor-deficient mice (M(3)R(-/-)) indicates that M3 receptors also regulate neutrophilic inflammation in response to cigarette smoke (CS). M(3) receptors are present on almost all cell types, and in this study we investigated the relative contribution of M(3) receptors on structural cells vs. inflammatory cells to CS-induced inflammation using bone marrow chimeric mice. Bone marrow chimeras (C56Bl/6 mice) were generated, and engraftment was confirmed after 10 wk. Thereafter, irradiated and nonirradiated control animals were exposed to CS or fresh air for four consecutive days. CS induced a significant increase in neutrophil numbers in nonirradiated and irradiated control animals (4- to 35-fold). Interestingly, wild-type animals receiving M(3)R(-/-) bone marrow showed a similar increase in neutrophil number (15-fold). In contrast, no increase in the number of neutrophils was observed in M3R(-/-) animals receiving wild-type bone marrow. The increase in keratinocyte-derived chemokine (KC) levels was similar in all smoke-exposed groups (2.5- to 5.0-fold). Microarray analysis revealed that fibrinogen-α and CD177, both involved in neutrophil migration, were downregulated in CS-exposed M(3)R(-/-) animals receiving wild-type bone marrow compared with CS-exposed wild-type animals, which was confirmed by RT-qPCR (1.6-2.5 fold). These findings indicate that the M(3) receptor on structural cells plays a proinflammatory role in CS-induced neutrophilic inflammation, whereas the M(3) receptor on inflammatory cells does not. This effect is probably not mediated via KC release, but may involve altered adhesion and transmigration of neutrophils via fibrinogen-α and CD177., (Copyright © 2015 the American Physiological Society.)

Cadherin-mediated cell-cell adhesion controls the morphology and function of epithelial cells and is a critical component of the pathology of chronic inflammatory disorders. Dynamic interactions between cadherins and the actin cytoskeleton are required for stable cell-cell contact. Besides actin, microtubules also target intercellular, cadherin-based junctions and contribute to their formation and stability. Here, we studied the role of microtubules in conjunction with Rho-like GTPases in the regulation of lung epithelial barrier function using real-time monitoring of transepithelial electrical resistance. Unexpectedly, we found that disruption of microtubules promotes epithelial cell-cell adhesion. This increase in epithelial barrier function is accompanied by the accumulation of beta-catenin at cell-cell junctions, as detected by immunofluorescence. Moreover, we found that the increase in cell-cell contact, induced by microtubule depolymerization, requires signaling through a RhoA/Rho kinase pathway. The Rac-1 GTPase counteracts this pathway, because inhibition of Rac-1 signaling rapidly promotes epithelial barrier function, in a microtubule- and RhoA-independent fashion. Together, our data suggest that microtubule-RhoA-mediated signaling and Rac-1 control lung epithelial integrity through counteracting independent pathways.

Administration of inhaled nitric oxide (iNO) is a potential therapeutic strategy to prevent bronchopulmonary dysplasia (BPD) in premature newborns with respiratory distress syndrome. We evaluated this approach in a rat model, in which premature pups were exposed to room air, hyperoxia, or a combination of hyperoxia and NO (8.5 and 17 ppm). We investigated the anti-inflammatory effects of prolonged iNO therapy by studying survival, histopathology, fibrin deposition, and differential mRNA expression (real-time RT-PCR) of key genes involved in the development of BPD. iNO therapy prolonged median survival 1.5 days (P = 0.0003), reduced fibrin deposition in a dosage-dependent way up to 4.3-fold (P < 0.001), improved alveolar development by reducing septal thickness, and reduced the influx of leukocytes. Analysis of mRNA expression revealed an iNO-induced downregulation of genes involved in inflammation (IL-6, cytokine-induced neutrophilic chemoattractant-1, and amphiregulin), coagulation, fibrinolysis (plasminogen activator inhibitor 1 and urokinase-type plasminogen activator receptor), cell cycle regulation (p21), and an upregulation of fibroblast growth factor receptor-4 (alveolar formation). We conclude that iNO therapy improves lung pathology and prolongs survival by reducing septum thickness, inhibiting inflammation, and reducing alveolar fibrin deposition in premature rat pups with neonatal hyperoxic lung injury.

The epithelial surface of the airways is the largest barrier-forming interface between the human body and the outside world. It is now well recognized that, at this strategic position, airway epithelial cells play an eminent role in host defense by recognizing and responding to microbial exposure. Conversely, inhaled microorganisms also respond to contact with epithelial cells. Our understanding of this cross talk is limited, requiring sophisticated experimental approaches to analyze these complex interactions. High-throughput technologies, such as DNA microarray analysis and serial analysis of gene expression (SAGE), have been developed to screen for gene expression levels at large scale within single experiments. Since their introduction, these hypothesis-generating technologies have been widely used in diverse areas such as oncology and brain research. Successful application of these genomics-based technologies has also revealed novel insights in host-pathogen interactions in both the host and pathogen. This review aims to provide an overview of the SAGE and microarray technology illustrated by their application in the analysis of host-pathogen interactions. In particular, the interactions between epithelial cells in the human lungs and clinically relevant microorganisms are the central focus of this review.

Secretory leukocyte proteinase inhibitor (SLPI) is a serine proteinase inhibitor that is produced locally in the lung by cells of the submucosal bronchial glands and by nonciliated epithelial cells. Its main function appears to be the inhibition of neutrophil elastase (NE). Recently, NE was found to enhance SLPI mRNA levels while decreasing SLPI protein release in airway epithelial cells. Furthermore, glucocorticoids were shown to increase both constitutive and NE-induced SLPI mRNA levels. In addition to NE, stimulated neutrophils also release alpha-defensins. Defensins are small, antimicrobial polypeptides that are found in high concentrations in purulent secretions of patients with chronic airway inflammation. Like NE, defensins induce interleukin-8 production in airway epithelial cells. This induction is sensitive to inhibition by the glucocorticoid dexamethasone and is prevented in the presence of alpha(1)-proteinase inhibitor. The aim of the present study was to investigate the effect of defensins on the production of SLPI and the related NE inhibitor elafin/SKALP in primary bronchial epithelial cells (PBECs). Defensins significantly increase SLPI protein release by PBECs in a time- and dose-dependent fashion without affecting SLPI mRNA synthesis. In the presence of alpha(1)-proteinase inhibitor, the defensin-induced SLPI protein release is further enhanced, but no effect was observed on SLPI mRNA levels. Dexamethasone did not affect SLPI protein release from control or defensin-treated PBECs. In addition, we observed a constitutive release of elafin/SKALP by PBECs, but this was not affected by defensins. The present results suggest a role for defensins in the dynamic regulation of the antiproteinase screen in the lung at sites of inflammation.