Intestinal Epithelial Cells Respond to Chronic Inflammation and Dysbiosis by Synthesizing H2O2

Abstract

The microbes in the gastrointestinal tract are separated from the host by a single layer of intestinal epithelial cells (IECs) that plays pivotal roles in maintaining homeostasis by absorbing nutrients and providing a physical and immunological barrier to potential pathogens. Preservation of homeostasis requires the crosstalk between the epithelium and the microbial environment. One epithelial-driven innate immune mechanism that participates in host-microbe communication involves the release of reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), toward the lumen. Phagocytes produce high amounts of ROS which is critical for microbicidal functions; the functional contribution of epithelial ROS, however, has been hindered by the lack of methodologies to reliably quantify extracellular release of ROS. Here, we used a modified Amplex Red assay to investigate the inflammatory and microbial regulation of IEC-generated H₂O₂ and the potential role of Duox2, a NADPH oxidase that is an important source of H₂O₂. We found that colonoids respond to interferon-γ and flagellin by enhancing production of H₂O₂ in a Duox2-mediated fashion. To extend these findings, we analyzed ex vivo production of H₂O₂ by IECs after acute and chronic inflammation, as well as after exposure to dysbiotic microbiota. While acute inflammation did not induce a significant increase in epithelial-driven H₂O₂, chronic inflammation caused IECs to release higher levels of H₂O₂. Furthermore, colonization of germ-free mice with dysbiotic microbiota from mice or patients with IBD resulted in increased H₂O₂ production compared with healthy controls. Collectively, these data suggest that IECs are capable of H₂O₂ production during chronic inflammation and dysbiotic states. Our results provide insight into luminal production of H₂O₂ by IECs as a read-out of innate defense by the mucosa.

Keywords: colitis; host-microbe interactions; inflammatory bowel disease; intestinal inflammation; microbiome.