Bacterial extracellular vesicles indirectly destabilize a human stem cell-derived blood-brain barrier on-chip through pro-inflammatory stimulation of immune cells

Technical Summary

Problem Statement
Pathogenic bacterial extracellular vesicles (BEVs) are known to disrupt the blood-brain barrier (BBB), contributing to neuroinflammation. However, prior in vitro investigations into this mechanism have relied on simplified models that may lack critical physiological components, specifically the immune system. This study addresses the gap in understanding whether BEVs directly compromise BBB integrity or if their disruptive effects are mediated indirectly through the stimulation of immune cells.

Methodology
The researchers utilized a sophisticated "lab-on-chip" model of the human BBB. This device was constructed using isogenic human induced pluripotent stem cell (hiPSC)-derived brain microvascular endothelial-like cells (BMECs) and pericytes. These cell types were separated by an ultrathin, porous silicon nitride membrane to mimic the physiological interface.

The experimental design involved two primary approaches:

1. Direct Exposure: The BBB-on-chip was treated directly with Escherichia coli-derived BEVs and free lipopolysaccharide (LPS) at physiological doses.
2. Indirect Exposure: THP-1 macrophages were first stimulated with BEVs to induce a pro-inflammatory response. The resulting conditioned media, containing cytokines and other secreted factors, was then applied to the BBB-on-chip model.

Key metrics for assessment included the expression of intercellular adhesion molecule-1 (ICAM-1) on BMEC surfaces, the integrity of tight junction proteins, and barrier permeability to small dye molecules.

Key Results

• Direct Effects: Treatment with BEVs or LPS successfully upregulated ICAM-1 on the BMEC surfaces, a marker associated with immune cell recruitment. However, at physiological doses, neither agent caused a pronounced loss of tight junction proteins nor increased the permeability of the barrier to small dye molecules.
• Indirect Effects: In contrast, when THP-1 macrophages were stimulated with BEVs, they produced elevated levels of pro-inflammatory cytokines. The conditioned media derived from these stimulated macrophages successfully disrupted BMEC tight junctions and significantly increased barrier permeability.

Key Contributions
This work provides evidence that the mechanism by which BEVs destabilize the BBB is not a direct interaction with endothelial cells but rather an indirect process mediated by immune activation. The study successfully demonstrates that simple co-culture models lacking an immune component may fail to capture the full pathological impact of BEVs on the BBB.

Significance
The paper concludes that incorporating an immune component is essential for accurately studying BEV-mediated disruption of BBB models. The findings suggest that previous models lacking immune cells may have underestimated the pathogenic potential of BEVs by missing the critical step of immune-mediated inflammation that ultimately compromises barrier integrity.