Supplementary Material for: Hydrogen Peroxide Is Crucial for NLRP3 Inflammasome-Mediated IL-1β Production and Cell Death in Pneumococcal Infections of Bronchial Epithelial Cells

Epithelial cells play a crucial role in detection of the pathogens as well as in initiation of the host immune response<i>. Streptococcus pneumoniae</i> (pneumococcus) is a typical colonizer of the human nasopharynx, which can disseminate to the lower respiratory tract and subsequently cause severe invasive diseases such as pneumonia, sepsis, and meningitis. Hydrogen peroxide (H₂O₂) is produced by pneumococci as a product of the pyruvate oxidase SpxB. However, its role as a virulence determinant in pneumococcal infections of the lower respiratory tract is not well understood. In this study, we investigated the role of pneumococcal-derived H₂O₂ in initiating epithelial cell death by analyzing the interplay between 2 key cell death pathways, namely, apoptosis and pyroptosis. We demonstrate that H₂O₂ primes as well as activates the NLRP3 inflammasome and thereby mediates IL-1β production and release. Furthermore, we show that pneumococcal H₂O₂ causes cell death via the activation of both apoptotic as well as pyroptotic pathways which are mediated by the activation of caspase-3/7 and caspase-1, respectively. However, H₂O₂-mediated IL-1β release itself occurs mainly via apoptosis.