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The respiratory epithelium, particularly the airway epithelium, is the primary infection site for respiratory pathogens. The apical surface of epithelial cells is constantly exposed to external stimuli including invading pathogens. Efforts have been made to establish organoid cultures to recapitulate the human respiratory tract. However, a robust and simple model with an easily accessible apical surface would benefit respiratory research. Here, we report the generation and characterization of apical-out airway organoids from the long-term expandable lung organoids that we previously established. The apical-out airway organoids morphologically and functionally recapitulated the human airway epithelium at a comparable level to the apical-in airway organoids. Moreover, apical-out airway organoids sustained productive and multicycle replication of SARS-CoV-2, and accurately recapitulated the higher infectivity and replicative fitness of the Omicron variants BA.5 and B.1.1.529 and an ancestral virus. In conclusion, we established a physiologically relevant and convenient apical-out airway organoid model for studying respiratory biology and diseases. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) structured organoids have become increasingly promising and effective in vitro models, and there is an urgent need for reliable models to assess health effects of inhaled pollutants on the human airway. In our study, we conducted a toxicity assessment of human airway organoids (hAOs) for tire wear particles (TWPs) as an emerging inhaled pollutant. We induced primary human bronchial epithelial cells (HBECs) to generated human airway organoids, which recapitulated the key features of human airway epithelial cells including basal cells, ciliated cells, goblet cells, and club cells. TWPs generated from the wearing of tire treads were considered a major source of emerging inhaled road traffic-derived non-exhaust particles, but their health effect on the lungs is poorly understood. We used human airway organoids to assess the toxicology of tire wear particles on the human airway. In an exposure study, the inhibitory effect of TWPs on the growth of human airway organoids was observed. TWPs induced significant cell apoptosis and oxidative stress in a dose-dependent manner. From the qPCR analysis, TWPs significantly up-regulated the expression pf genes involved in the inflammation response. Additionally, the exposure of TWPs reduced SCGB1A1 gene expression associated with the function of the club cell and KRT5 gene expression related to the function of basal cells. In conclusion, this was first study using human airway organoids for a toxicological assessment of TWPs, and our findings revealed that human airway organoids provide an evaluation model of inhaled pollutants potentially affecting the lungs.

The numerous global outbreaks and continuous reassortments of highly pathogenic avian influenza (HPAI) A(H5N6/H5N8) clade 2.3.4.4 viruses in birds pose a major risk to the public health. We investigated the tropism and innate host responses of 5 recent HPAI A(H5N6/H5N8) avian isolates of clades 2.3.4.4b, e, and h in human airway organoids and primary human alveolar epithelial cells. The HPAI A(H5N6/H5N8) avian isolates replicated productively but with lower competence than the influenza A(H1N1)pdm09, HPAI A(H5N1), and HPAI A(H5N6) isolates from humans in both or either models. They showed differential cellular tropism in human airway organoids; some infected all 4 major epithelial cell types: ciliated cells, club cells, goblet cells, and basal cells. Our results suggest zoonotic potential but low transmissibility of the HPAI A(H5N6/H5N8) avian isolates among humans. These viruses induced low levels of proinflammatory cytokines/chemokines, which are unlikely to contribute to the pathogenesis of severe disease.

The respiratory epithelium, particularly the airway epithelium, is the primary infection site for respiratory pathogens. The apical surface of epithelial cells is constantly exposed to external stimuli including invading pathogens. Efforts have been made to establish organoid cultures to recapitulate the human respiratory tract. However, a robust and simple model with an easily accessible apical surface would benefit respiratory research. Here, we report the generation and characterization of apical-out airway organoids from the long-term expandable lung organoids that we previously established. The apical-out airway organoids morphologically and functionally recapitulated the human airway epithelium at a comparable level to the apical-in airway organoids. Moreover, apical-out airway organoids sustained productive and multicycle replication of SARS-CoV-2, and accurately recapitulated the higher infectivity and replicative fitness of the Omicron variants BA.5 and B.1.1.529 and an ancestral virus. In conclusion, we established a physiologically relevant and convenient apical-out airway organoid model for studying respiratory biology and diseases.

The numerous global outbreaks and continuous reassortments of highly pathogenic avian influenza (HPAI) A(H5N6/H5N8) clade 2.3.4.4 viruses in birds pose a major risk to the public health. We investigated the tropism and innate host responses of 5 recent HPAI A(H5N6/H5N8) avian isolates of clades 2.3.4.4b, e, and h in human airway organoids and primary human alveolar epithelial cells. The HPAI A(H5N6/H5N8) avian isolates replicated productively but with lower competence than the influenza A(H1N1)pdm09, HPAI A(H5N1), and HPAI A(H5N6) isolates from humans in both or either models. They showed differential cellular tropism in human airway organoids; some infected all 4 major epithelial cell types: ciliated cells, club cells, goblet cells, and basal cells. Our results suggest zoonotic potential but low transmissibility of the HPAI A(H5N6/H5N8) avian isolates among humans. These viruses induced low levels of proinflammatory cytokines/chemokines, which are unlikely to contribute to the pathogenesis of severe disease. [ABSTRACT FROM AUTHOR]

Despite significant advances in understanding the general health impacts of air pollution, the toxic effects of air pollution on cells in the human respiratory tract are still elusive. A robust, biologically relevant in vitro model for recapitulating the physiological response of the human airway is needed to obtain a thorough understanding of the molecular mechanisms of air pollutants. In this study, by using 1-nitropyrene (1-NP) as a proof-of-concept, we demonstrate the effectiveness and reliability of evaluating environmental pollutants in physiologically active human airway organoids. Multimodal imaging tools, including live cell imaging, fluorescence microscopy, and MALDI-mass spectrometry imaging (MSI), were implemented to evaluate the cytotoxicity of 1-NP for airway organoids. In addition, lipidomic alterations upon 1-NP treatment were quantitatively analyzed by nontargeted lipidomics. 1-NP exposure was found to be associated with the overproduction of reactive oxygen species (ROS), and dysregulation of lipid pathways, including the SM-Cer conversion, as well as cardiolipin in our organoids. Compared with that of cell lines, a higher tolerance of 1-NP toxicity was observed in the human airway organoids, which might reflect a more physiologically relevant response in the native airway epithelium. Collectively, we have established a novel system for evaluating and investigating molecular mechanisms of environmental pollutants in the human airways via the combinatory use of human airway organoids, multimodal imaging analysis, and MS-based analyses.

Three-dimensional (3D) structured organoids have become increasingly promising and effective in vitro models, and there is an urgent need for reliable models to assess health effects of inhaled pollutants on the human airway. In our study, we conducted a toxicity assessment of human airway organoids (hAOs) for tire wear particles (TWPs) as an emerging inhaled pollutant. We induced primary human bronchial epithelial cells (HBECs) to generated human airway organoids, which recapitulated the key features of human airway epithelial cells including basal cells, ciliated cells, goblet cells, and club cells. TWPs generated from the wearing of tire treads were considered a major source of emerging inhaled road traffic-derived non-exhaust particles, but their health effect on the lungs is poorly understood. We used human airway organoids to assess the toxicology of tire wear particles on the human airway. In an exposure study, the inhibitory effect of TWPs on the growth of human airway organoids was observed. TWPs induced significant cell apoptosis and oxidative stress in a dose-dependent manner. From the qPCR analysis, TWPs significantly up-regulated the expression pf genes involved in the inflammation response. Additionally, the exposure of TWPs reduced SCGB1A1 gene expression associated with the function of the club cell and KRT5 gene expression related to the function of basal cells. In conclusion, this was first study using human airway organoids for a toxicological assessment of TWPs, and our findings revealed that human airway organoids provide an evaluation model of inhaled pollutants potentially affecting the lungs., 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 © 2023 Jiang, Lu, Du, Li, Cheng, Bi, Li, Zhuang, Ren, Wang and Ji.)