Modeling Clostridioides difficile toxin pathogenesis and antiserum protection in an immunocompetent intestine-on-chip platform.

Clostridioides difficile (C. difficile) is a leading cause of nosocomial diarrhea and colitis, including severe pseudomembranous colitis, particularly following antibiotic-induced dysbiosis. The pathogenesis of C. difficile infection (CDI) is primarily driven by the action of two large exotoxins, toxin A (TcdA) and toxin B (TcdB), which compromise intestinal epithelial integrity and trigger strong mucosal inflammation. These toxins lead to the disassembly of epithelial junctions, immune cell infiltration, and release of pro-inflammatory mediators. Despite extensive research, mechanistic insight into C. difficile-host interactions and correlates of protection remain limited, in part due to the physiological constraints of conventional two-dimensional (2D) in vitro models. Here, we present a three-dimensional (3D) microphysiological Intestine-on-Chip (IoC) model as a dynamic and immunocompetent in vitro platform to study toxin-mediated pathogenesis and therapeutic interventions in CDI. In contrast to traditional static cell culture systems composed solely of epithelial monolayers, the immunocompetent IoC (i-IoC) model integrates Caco-2 C2BBe1 epithelial cells, primary human umbilical vein endothelial cells (HUVECs), monocyte-derived macrophages, and circulating neutrophils (polymorphonuclear leukocytes, (PMN)) under continuous perfusion, thus more closely mimicking the tissue architecture and immune microenvironment of the human intestine. Upon stimulation with purified TcdA and TcdB, the i-IoC model exhibited toxin-specific disruption of epithelial junctional proteins, macrophage depletion, elevated cytokine secretion, and recruitment and transmigration of PMN, thereby replicating hallmark features of acute CDI. Notably, the model responded with higher sensitivity and biological complexity than static 2D cultures. Toxin-neutralizing antibody sera effectively attenuated these pathological responses, reducing both structural damage and inflammatory mediator release. Our findings demonstrate that the i-IoC model faithfully recapitulates key aspects of CDI pathophysiology, including epithelial damage, immune cell dynamics, and cytokine-driven inflammation. This platform offers a versatile and translationally relevant tool to study host-pathogen interactions and to evaluate preventive or therapeutic strategies aimed at mitigating C. difficile toxin (CDT)-mediated tissue injury.