Shiga Toxin Induces Apoptosis <i>via</i> ROS–Caspase Activation in Human Cerebral Endothelial Cell Line hCMEC/D3 and Astrocyte Co-Culture

Hemolytic uremic syndrome (HUS), a fatal complication of Shiga toxin-producing <i>Escherichia coli</i> (STEC) infection, is classically characterized by acute renal failure, but frequently accompanied by central nervous system (CNS) dysfunction. Because the CNS is normally protected by the blood-brain barrier (BBB), the toxin-mediated BBB injury is considered to be a major cause of neurologic sequelae in STEC infection. Here, we delineate how Shiga toxin type 1a (Stx1a) and Shiga toxin type 2a (Stx2a) compromise BBB-like endothelial barrier integrity with the human brain microvascular endothelial cell line hCMEC/D3 as an <i>in vitro</i> model, complemented by an endothelial-astroglial co-culture system. Stx1a/Stx2a exposure induced the MAPK pathway and ER stress, triggering caspase-mediated apoptosis and pro-inflammatory cytokines expression. Coincidentally, permeability across tight junctions was impaired, with junctional protein loss and increased paracellular permeability. Pharmacologic inhibition of caspases prevented cytotoxicity and tight junction loss, indicating a role for the apoptotic process in barrier breach. In co-cultures with transwell, human astrocytes (A172) demonstrated caspase activity and cytokine induction even without direct exposure to toxins, indicating endothelial injury-release paracrine activity in the propagation of BBB injury. Reactive oxygen species (ROS) also accumulated distal to toxin exposure and aligned with apoptotic and barrier phenotypes, indicating a ROS-caspase pathway in endothelial cell injury. Collectively, our findings show that Stx may impair BBB integrity through ROS accumulation and caspase-dependent apoptosis. This is the first study establishing hCMEC/D3 cells as a model for elucidating Stx-induced BBB disruption, providing mechanistic insights for the therapeutic development against CNS complications in HUS.