Dual Oxygen-Partitioned Co-Culture Uncovers Microbe-Specific Epithelial Stress and Homeostatic Programs

Abstract Direct epithelial-microbial interactions occur across a steep aerobic-anaerobic interface in the intestine, yet mechanistic analysis has been limited by the difficulty of sustaining oxygen-dependent epithelial cells together with strictly anaerobic microbes in vitro . Here, we establish a dual oxygen-partitioned co-culture system that reproducibly maintains aerobic intestinal epithelial cells and obligate anaerobic gut bacteria, enabling controlled analysis of epithelial responses under physiologically relevant oxygen architecture. Using Mediterraneibacter gnavus and Lacticaseibacillus casei as contrasting microbial partners, we identify distinct epithelial programs. M. gnavus induces epithelial stress characterized by tight-junction disruption, suppression of mitochondrial respiration, and activation of chromatin-associated and innate immune regulatory pathways, whereas L. casei preserves barrier integrity and supports mitochondrial capacity with restrained immune modulation. Integrated transcriptomic and proteomic analyses reveal a shared epithelial energy-conserving response to microbial proximity, upon which microbe-specific stress or homeostatic programs are imposed. In vivo analysis using a dextran sodium sulfate-induced colitis model demonstrates that these epithelial programs become physiologically relevant under inflammatory conditions, with L. casei promoting epithelial repair and immune rebalancing. Together, these findings define oxygen-partitioned epithelial-microbial interactions as a determinant of microbe-specific epithelial states.