Human Primary Cell-Based In Vitro Blood-Brain Barrier to Study Toxoplasma Gondii Brain Invasion and Drug Screening

Abstract

Background: Toxoplasmosis is one of the most widespread parasitic infections that affects approximately one-third of the human population and nearly 80% of the Lebanese population. It is caused by Toxoplasma gondii (T. gondii), an obligate intracellular protozoan parasite capable of crossing the blood-brain barrier (BBB) and forming cysts within the brain, leading to chronic toxoplasmosis (CT). CT can be associated with various neuropsychiatric behavioral disorders, primary neuropathies, brain tumors, and its reactivation is a life-threatening condition in immunocompromised patients. Despite advances in pharmacology, effective treatments for CT remain a significant challenge, primarily due to the selective permeability nature of the BBB, which restricts the delivery of most drugs into the central nervous system. Therefore, to address this issue, a physiologically relevant in vitro BBB model is essential to study the barrier selectivity and functionality, and to elucidate the mechanisms of T. gondii brain invasion, ultimately supporting the development of effective therapeutic strategies.

Aim: Our study seeks to develop a biomimetic in vitro BBB model using primary human aortic endothelial cells (HAECs) and Normal human astrocytes (NHAs), which are the key components of the neurovascular unit that forms the BBB. To enhance structural and functional integrity, the model incorporates porcine brain-derived extracellular matrix (ECM). This model will enable us to investigate T. gondii brain invasion and will facilitate the screening for potential therapeutic drugs.

Methods: The endothelial barrier was established under three different conditions: uncoated, Matrigel-coated, and Matrigel combined with porcine-derived brain ECM. Functional and molecular assays were conducted to evaluate barrier integrity, including trans-endothelial electric resistance (TEER) and permeability assays using fluorescent tracers such as Sodium fluorescein and Evans’ Blue. Additionally, gene and protein expression analysis of tight junction markers such as ZO-1, Cx43, and CLDN-5 was performed using quantitative qRT-PCR, western blot, and immunofluorescence (IF). The pathway of tachyzoite dissemination through the endothelial barrier was visualized by live-imaging assay and further confirmed by transmigration assay of infected versus control THP-1 cells.

Results: Culturing of endothelial cells on brain ECM enhanced the expression of brain-specific tight junction proteins, including ZO-1 and CLDN-5. This upregulation improved barrier integrity, as evidenced by the significant increase in TEER values, in The brain ECM-based model was compared to both control and Matrigel conditions. Additionally, this model exhibited reduced permeability of some molecular tracers, underscoring the critical role of brain ECM in enhancing the functional properties of the barrier. For T. gondii results, the imaging revealed that tachyzoites utilize both the transcellular and "Trojan horse" mechanisms for crossing the barrier, with a marked predominance of the latter.

Conclusion: Developing an in vitro blood-brain barrier BBB model represents a vital step toward understanding the neuropathology of infections caused by T. gondii. Such models provide valuable insight into the parasite mechanisms of brain invasion and offer a promising platform for therapeutic development, while minimizing the use of in vivo models.