Examining the Effects of Physiological Gases on Intestinal Barrier Integrity and Function in a Human Enteroid Model System

Abstract

Warfighters are commonly exposed to unique physiologic stressors that affect their physical and mental performance, as well as their overall health. Hypoxia has been shown to induce oxidative stress, inflammation leading to intestinal barrier disruption and is recognized as a potential stressor affecting the warfighter. Similarly, an excess of carbon dioxide (CO2), or hypercapnia, also affects intestinal tissue, as levels of CO2 are inversely coupled with the level of oxygen and are found to be elevated in hypoxic tissue. The GI tract is thought to have a low-grade, albeit constant, level of inflammation and is home to multiple immune cell types. In addition, both epithelial and immune cells in the intestine can sense and respond to various stimuli including pathogens, and compromises to intestinal barrier integrity due to injury or stress can result. In this study, we sought to determine the extent to which hypoxia and hypercapnia degrade intestinal cellular integrity through inflammation-mediated mechanisms. Intestinal epithelial organoids (enteroids) derived from the duodenum were exposed to hypoxic (0.5% O2) or hypercapnic (20% CO2) conditions for a period of 72 hours. Initial results indicated intestinal barrier integrity was compromised in hypoxia and 20% CO2 conditions as evidenced by transepithelial electrical resistance (TEER), with hypoxia (0.7 Ω) and 20% CO2 (0.85 Ω) being significantly lower than control (1.19 Ω; P < 0.05). Further, immunofluorescence staining of several tight-junction proteins, including Claudin-2, -3, -7, Zonula occludens-1, and Occludin, show disintegration of these proteins under hypoxic and hypercapnic stress. Scanning Electron Microscopy revealed microvilli-like structures are shortened under hypercapnic stress, while hypoxic conditions showed cells with compromised structure and detachment from membranes. Preliminary gene expression results suggest alterations in tight-junction markers as well as in genes involved in inflammatory response pathways. In addition, IL-8, a macrophage-mediated chemokine produced in intestinal epithelial cells and involved in the innate immune response, was found to be significantly downregulated in hypoxia (58.7 pg/mL) and hypercapnia (49.7 pg/mL) compared to control (190.7 pg/mL; P < 0.05). In sum, our data indicate hypoxic and hypercapnic conditions lead to alterations in molecules involved in the innate immune response pathway, tight-junction markers, as well as compromised epithelial barrier integrity. Future experiments will be aimed at identifying the mechanism(s) responsible for diminished intestinal barrier function as well as identify potential preventative strategies.