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Phosphodiesterase (PDE) inhibition has been identified in animal studies as a new treatment option for neonatal lung injury, and as potentially beneficial for early lung development and function. However, our group could show that the inhaled PDE4 inhibitor GSK256066 could have dose-dependent detrimental effects and promote lung inflammation in the premature lung. In this study, the effects of a high and a low dose of GSK256066 on lung function, structure and alveolar development were investigated. In a triple hit lamb model of Ureaplasma-induced chorioamnionitis, prematurity, and mechanical ventilation, 21 animals were treated as unventilated (NOVENT) or 24 h ventilated controls (Control), or with combined 24 h ventilation and low dose (iPDE1) or high dose (iPDE10) treatment with inhaled GSK 256066. We found that high doses of an inhaled PDE4 inhibitor impaired oxygenation during mechanical ventilation. In this group, the budding of secondary septae appeared to be decreased in the preterm lung, suggesting altered alveologenesis. Ventilation-induced structural and functional changes were only modestly ameliorated by a low dose of PDE4 inhibitor. In conclusion, our findings indicate the narrow therapeutic window of PDE4 inhibitors in the developing lung. [ABSTRACT FROM AUTHOR]

The culture of lung organoids relies on drops of basement membrane matrices. This comes with limitations, for example, concerning the microscopic monitoring and imaging of the organoids in the drops. Also, the culture technique is not easily compatible with micromanipulations of the organoids. In this study, we investigated the feasibility of the culture of human bronchial organoids in defined x-, y- and z-positions in a polymer film-based microwell array platform. The circular microwells have thin round/U-bottoms. For this, single cells are first precultured in drops of basement membrane extract (BME). After they form cell clusters or premature organoids, the preformed structures are then transferred into the microwells in a solution of 50% BME in medium. There, the structures can be cultured toward differentiated and mature organoids for several weeks. The organoids were characterized by bright-field microscopy for size growth and luminal fusion over time, by scanning electron microscopy for overall morphology, by transmission electron microscopy for the existence of microvilli and cilia, by video microscopy for beating cilia and swirling fluid, by live-cell imaging, by fluorescence microscopy for the expression of cell-specific markers and for proliferating and apoptotic cells, and by ATP measurement for extended cell viability. Finally, we demonstrated the eased micromanipulation of the organoids in the microwells by the example of their microinjection., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Roman Truckenmueller reports a relationship with 300MICRONS GmbH that includes: board membership and equity or stocks. Stefan Giselbrecht reports a relationship with 300MICRONS GmbH that includes: board membership and equity or stocks., (© 2023 The Authors.)

Perinatal inflammatory stress is strongly associated with adverse pulmonary outcomes after preterm birth. Antenatal infections are an essential perinatal stress factor and contribute to preterm delivery, induction of lung inflammation and injury, pre-disposing preterm infants to bronchopulmonary dysplasia. Considering the polymicrobial nature of antenatal infection, which was reported to result in diverse effects and outcomes in preterm lungs, the aim was to examine the consequences of sequential inflammatory stimuli on endogenous epithelial stem/progenitor cells and vascular maturation, which are crucial drivers of lung development. Therefore, a translational ovine model of antenatal infection/inflammation with consecutive exposures to chronic and acute stimuli was used. Ovine fetuses were exposed intra-amniotically to Ureaplasma parvum 42 days (chronic stimulus) and/or to lipopolysaccharide 2 or 7 days (acute stimulus) prior to preterm delivery at 125 days of gestation. Pulmonary inflammation, endogenous epithelial stem cell populations, vascular modulators and morphology were investigated in preterm lungs. Pre-exposure to UP attenuated neutrophil infiltration in 7d LPS-exposed lungs and prevented reduction of SOX-9 expression and increased SP-B expression, which could indicate protective responses induced by re-exposure. Sequential exposures did not markedly impact stem/progenitors of the proximal airways (P63+ basal cells) compared to single exposure to LPS. In contrast, the alveolar size was increased solely in the UP+7d LPS group. In line, the most pronounced reduction of AEC2 and proliferating cells (Ki67+) was detected in these sequentially UP + 7d LPS-exposed lambs. A similar sensitization effect of UP pre-exposure was reflected by the vessel density and expression of vascular markers VEGFR-2 and Ang-1 that were significantly reduced after UP exposure prior to 2d LPS, when compared to UP and LPS exposure alone. Strikingly, while morphological changes of alveoli and vessels were seen after sequential microbial exposure, improved lung function was observed in UP, 7d LPS, and UP+7d LPS-exposed lambs. In conclusion, although sequential exposures did not markedly further impact epithelial stem/progenitor cell populations, re-exposure to an inflammatory stimulus resulted in disturbed alveolarization and abnormal pulmonary vascular development. Whether these negative effects on lung development can be rescued by the potentially protective responses observed, should be examined at later time points., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Widowski, Reynaert, Ophelders, Hütten, Nikkels, Severens-Rijvers, Cleutjens, Kemp, Newnham, Saito, Usuda, Payne, Jobe, Kramer, Delhaas and Wolfs.)