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

## TL;DR

Shiga toxin from E. coli causes brain barrier damage by triggering cell death and inflammation, leading to neurological issues in HUS.

## Contribution

This is the first study using hCMEC/D3 cells to model Shiga toxin-induced blood-brain barrier disruption.

## Key findings

- Shiga toxins Stx1a and Stx2a trigger apoptosis and inflammation in brain endothelial cells.
- Pharmacologic caspase inhibition prevents toxin-induced barrier damage and cell death.
- Reactive oxygen species (ROS) and caspase pathways are central to toxin-induced endothelial injury.

## Abstract

Hemolytic uremic syndrome (HUS), a fatal complication of Shiga toxin-producing Escherichia coli (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 in vitro 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.