Growing evidence links prenatal exposure to particulate matter (PM2.5) with reduced lung function and incidence of pulmonary diseases in infancy and childhood. However, the underlying biological mechanisms of how prenatal PM2.5 exposure affects the lungs are incompletely understood, which explains the lack of an ideal in vitro lung development model. Human pluripotent stem cells (hPSCs) have been successfully employed for in vitro developmental toxicity evaluations due to their unique ability to differentiate into any type of cell in the body. In this study, we investigated the developmental toxicity of diesel fine PM (dPM2.5) exposure during hPSC-derived alveolar epithelial cell (AEC) differentiation and three-dimensional (3D) multicellular alveolar organoid (AO) development. We found that dPM2.5 (50 and 100 μg/mL) treatment disturbed the AEC differentiation, accompanied by upregulation of nicotinamide adenine dinucleotide phosphate oxidases and inflammation. Exposure to dPM2.5 also promoted epithelial-to-mesenchymal transition during AEC and AO development via activation of extracellular signal-regulated kinase signaling, while dPM2.5 had no effect on surfactant protein C expression in hPSC-derived AECs. Notably, we provided evidence, for the first time, that angiotensin-converting enzyme 2, a receptor to mediate the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) entry into target cells, and the cofactor transmembrane protease serine 2 were significantly upregulated in both hPSC-AECs and AOs treated with dPM2.5. In conclusion, we demonstrated the potential alveolar development toxicity and the increase of SARS-Cov-2 susceptibility of PM2.5. Our findings suggest that an hPSC-based 2D and 3D alveolar induction system could be a useful in vitro platform for evaluating the adverse effects of environmental toxins and for virus research.
Human pluripotent stem cell (hPSC)-derived alveolar epithelial cells (AECs) provide new opportunities for understanding lung development and the treatment of pulmonary diseases. However, toxicity assessments using hPSC-AECs have not been undertaken. In this study, we generated functional AECs from hPSCs and evaluated their inflammatory and apoptotic responses to cadmium (Cd) exposure (1, 5, and 10 μM) for 24 h compared with the human bronchial epithelial cell line (BEAS-2B) and primary AECs as controls. Our data showed that Cd (10 μM) treatment induced substantial inflammatory responses and apoptosis in BEAS-2B cells, but not in both hPSC-AECs and primary AECs. Interestingly, conditioned medium from AEC cultures significantly alleviated apoptotic and inflammatory responses to Cd exposure in BEAS-2B cells. Using cytokine arrays, several potential factors secreted from hPSC-AECs and primary AECs were detected and may be involved in reducing Cd-induced cytotoxicity. We also observed higher expression of surfactant proteins B and C in both hPSC-AECs and primary AECs, which may contribute to protection against Cd-induced cytotoxicity. These results suggested that hPSC-AECs phenotypically and functionally resemble primary AECs and could be more biologically relevant alternatives for evaluating the pathological contribution of confirmed or potential pulmotoxic materials included in smoking and microdust.
Inflammasomes, which are intracellular sensors of endogenous or exogenous danger signals, activate caspase-1, resulting in interleukin (IL)-1β maturation. Although most studies on inflammasomes have been performed in human and/or mouse-derived macrophages, porcine inflammasome activation has not been elucidated even though pigs are considered one of the best animal models for translational and preclinical investigations. In this study, we optimized detection of porcine IL-1β secretion, which is the most well established indicator of inflammasome activation, and compared inflammasome activation between miniature and domestic pigs as well as between porcine and murine macrophages. In our results, anti-sera against murine IL-1β had higher affinity to porcine IL-1β than anti-sera against human IL-1β, even though the amino acid sequence of porcine IL-1β was more similar to that of human IL-1β. In addition, there was no significant difference in inflammasome activation between miniature and domestic pigs. Furthermore, well established inflammasome triggers (ATP, nigericin, and crystals) in humans and mice had similar effects on porcine NLRP3 inflammasome activation. We further elucidated the upstream signaling pathway of porcine inflammasome activation using pharmacological inhibitors. Similar to the mechanisms of inflammasome activation in humans and mice, potassium efflux and reactive oxygen species generation were confirmed as key pathways in porcine inflammasome activation. Thus, inflammasome activation in pigs is not different from that in humans or mice.
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