Intestinal Epithelial Cells Respond to Chronic Inflammation and Dysbiosis by Synthesizing HO.

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 (HO), 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 HO and the potential role of Duox2, a NADPH oxidase that is an important source of HO. We found that colonoids respond to interferon-γ and flagellin by enhancing production of HO in a Duox2-mediated fashion. To extend these findings, we analyzed production of HO 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 HO, chronic inflammation caused IECs to release higher levels of HO. Furthermore, colonization of germ-free mice with dysbiotic microbiota from mice or patients with IBD resulted in increased HO production compared with healthy controls. Collectively, these data suggest that IECs are capable of HO production during chronic inflammation and dysbiotic states. Our results provide insight into luminal production of HO by IECs as a read-out of innate defense by the mucosa.