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Int J Biol Sci 2023; 19(6):1875-1893. doi:10.7150/ijbs.80605 This issue Cite

Research Paper

Hong Zhou†, Qun Zhang†, Wen Huang, Shulan Zhou, Yanli Wang, Xiaoning Zeng, Hong Wang, Weiping Xie , Hui Kong

Department of Pulmonary & Critical Care Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China†These authors contributed equally to this work.

✉ Corresponding authors: Hui Kong, M.D., Ph.D., Email: konghuiedu.cn. Weiping Xie, M.D., Ph.D., Email: wpxieedu.cn. Department of Pulmonary & Critical Care Medicine, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu 210029, P.R. China. Tel: +86-25-68136426; Fax: +86-25-68136269. More

Silica-induced lung epithelial injury and fibrosis are vital pathogeneses of silicosis. Although the NOD-like receptor protein 3 (NLRP3) inflammasome contributes to silica-induced chronic lung inflammation, its role in epithelial injury and regeneration remains unclear. Here, using mouse lung stem/progenitor cell-derived organotypic systems, including 2D air-liquid interface and 3D organoid cultures, we investigated the effects of the NLRP3 inflammasome on airway epithelial phenotype and function, cellular injury and regeneration, and the potential mechanisms. Our data showed that silica-induced NLRP3 inflammasome activation disrupted the epithelial architecture, impaired mucociliary clearance, induced cellular hyperplasia and the epithelial-mesenchymal transition in 2D culture, and inhibited organoid development in 3D system. Moreover, abnormal expression of the stem/progenitor cell markers SOX2 and SOX9 was observed in the 2D and 3D organotypic models after sustained silica stimulation. Notably, these silica-induced structural and functional abnormalities were ameliorated by MCC950, a selective NLRP3 inflammasome inhibitor. Further studies indicated that the NF-κB, Shh-Gli and Wnt/β-catenin pathways were involved in NLRP3 inflammasome-mediated abnormal differentiation and dysfunction of the airway epithelium. Thus, prolonged NLRP3 inflammasome activation caused injury and aberrant lung epithelial regeneration, suggesting that the NLRP3 inflammasome is a pivotal target for regulating tissue repair in chronic inflammatory lung diseases.

Keywords: NLRP3 inflammasome, Lung stem/progenitor cells (LSPCs), Organoids, Repair, Regeneration

The occurrence of chronic inflammation in the lung due to various insults is a pivotal etiology for the development of chronic obstructive pulmonary disease (COPD), corona virus disease 2019 (COVID-19), idiopathic pulmonary fibrosis (IPF), occupational pulmonary disease, and other conditions [1-4]. Silicosis, an incurable and progressive pulmonary disease, is characterized by persistent inflammation and irreversible fibrosis caused by the inhalation of silica particles [5, 6]. Accumulating evidence supports the observation that sustained damage to the distal airway epithelium due to silica is the dominant cause of airway structure remodeling and lung fibrosis in silicosis [7]. Histologically, terminal bronchioles are particularly susceptible to silica particles, which cause mucous plugging [8], epithelial dysfunction [9, 10] and ultimately peribronchiolar fibrosis, bronchiolar obstruction or obliteration [11, 12].

Regarding pulmonary fibrotic diseases, previous studies have reinforced the importance of macrophages, fibroblasts and epithelial cells in maintaining the structural and functional integrity of the lung [13-16]. However, recent studies suggest that lung stem/progenitor cells (LSPCs) play an important role in lung tissue injury, repair and regeneration [17, 18]. Increasing experimental evidence has revealed that epithelial progenitor cell injury and aberrant repair lead to fibrosis and bronchiolitis obliterans-like conditions [19]. With repeated exposure to silica particles, LSPC proliferation and differentiation are impaired during the development of silicosis [10]. In IPF, ongoing injurious stimuli induce an aberrant environment that drives LSPCs away from terminal differentiation and initiates a profibrotic cascade [20]. In addition, distal airway stem/progenitor cells could ameliorate bleomycin-induced pulmonary fibrosis by promoting lung regeneration [21].

The NLRP3 inflammasome is a widely distributed cytosolic multiprotein complex comprising NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD domain (ASC), and pro-Caspase-1. The assembled NLRP3 inflammasome promotes pro-Caspase-1 maturation and converts pro‐interleukin‐1β (IL‐1β) and pro‐IL‐18 into their bioactive forms [22, 23]. To date, the NLRP3 inflammasome has been reported to play a central role in several inflammatory respiratory diseases, such as asthma [24], sarcoidosis [25], cystic fibrosis [26], acute lung injury [27] and COVID-19 [28, 29]. In addition, silica is a well-recognized activator of the NLRP3 inflammasome, which induces inflammatory damage and mesenchymal transition of airway epithelial cells, thereafter promoting pulmonary fibrosis in silicosis [30, 31]. However, whether silica-induced NLRP3 inflammasome activation mediates LSPC injury, repair and regeneration remains unclear.

Lung organotypic models are stem/progenitor cell-derived and self-organizing structures. They simulate the morphogenesis, structures and functions of the respiratory system in vivo, providing a versatile and reliable in vitro culture system to study lung development and regenerative medicine, model diseases and screen drugs [32, 33]. During the past decade, lung organotypic models, including 2D air-liquid interface (ALI) and 3D organoid cultures, have become remarkable platforms to investigate the differentiation and repair of LSPCs, morphogenesis and function of ciliated epithelium, as well as mucus production and airway clearance [34, 35]. Recently, important advances in illustrating the mechanisms of COVID-19 have been reported using organoid platforms [36, 37]. In this study, we employed mouse LSPC-derived lung epithelial organotypic models (including ALI and organoid cultures) to unveil the effects and mechanisms of lung epithelial injury and regeneration in response to sustained silica stimulation. We discovered the critical role of NLRP3 inflammasome activation in enhancing silica-induced lung epithelial injury and aberrant repair, providing a promising therapeutic target for inhalable particle-related lung injuries and diseases.

An airway epithelium-like structure from the lungosphere-dissociated LSPCs was successfully generated in our ALI culture system, as indicated by the formation of pseudostratified columnar ciliated epithelium-containing MUC5AC+ goblet cells (Figures S1 and S2). As shown in Figures 1A and 1B, both the immunohistochemistry and western blot results for the components and products of the NLRP3 inflammasome (including NLRP3, pro-Caspase-1, ASC, Caspase-1 p20 and IL-1β) confirmed that sustained silica stimulation resulted in significant activation of the NLRP3 inflammasome in the LSPC-differentiated airway epithelium, which was blocked by MCC950, a selective NLRP3 inflammasome inhibitor. Notably, routine hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and Masson's trichrome staining showed that sustained stimulation with silica for 28 days led to thickened and disorganized LSPC-differentiated airway epithelium that exhibited irregular surfaces, large cavities, overproduction of mucus, and accumulation of collagen fibers (Figures 1C-F). A single treatment with MCC950 alone had no significant effects on the generation of the airway epithelium, while it prevented the epithelial disorganization induced by silica (Figure 1C).

Pyroptosis is a specific programmed cell death mediated by the activated NLRP3 inflammasome that is involved in various respiratory diseases, including pulmonary fibrosis [30, 38]. Once activated, the NLRP3 inflammasome downstream of Caspase-1 cleaves cytoplasmic Gasdermin D (GSDMD) to release its N-terminal domain, thereafter forming membrane pores to induce pyroptotic cell death [39]. As shown in the western blot analysis, compared to that of the control group, silica treatment significantly increased the levels of GSDMD and the GSDMD N-terminal compared with the control group, changes that were reversed by the NLRP3 inflammasome inhibitor MCC950 (Figure 2A). Consistently, the immunofluorescence staining showed that sustained silica stimulation resulted in a greater number of membrane-distributed GSDMD+ cells in LSPC-derived airway epithelial cells in the ALI model, which was inhibited by MCC950 (Figure 2B).

Effects of silica-induced NLRP3 inflammasome activation on the disorganization of the LSPC-derived airway epithelium in the ALI model. (A) Representative photographs of immunohistochemistry for NLRP3, Caspase-1 and IL-1β staining in the z-stack of ALI cultures. Scale bar, 20 μm. (B) Representative images and western blot analysis of NLRP3, pro-Caspase-1, ASC and Caspase-1 p20 in ALI cultures. MCC950 markedly decreased silica-induced NLRP3 inflammasome activation in the LSPC-derived airway epithelium (n=3-4). Data are presented as the mean ± standard error of the mean (SEM): *P