Blood Serum Cytokines Induce Changes in Blood‐Brain Barrier Permeability and Gene Expression in an In Vitro Blood‐Brain Barrier Model

Abstract

The blood‐brain barrier (BBB), a semi‐permeable interface between the brain and the blood, is a key player in the of development and progression of brain‐related illnesses such as traumatic brain injury (TBI), Alzheimer’s disease (AD), multiple sclerosis (MS), and schizophrenia (SCZ). With these disorders, pathologic BBB leakiness is caused by changes in the cellular milieu on the brain‐side as well as on the blood‐side of the BBB. On the brain‐side, injury to neurons, astrocytes, microglia, and pericytes can lead to increased BBB leakiness, while on the blood‐side, pathophysiologic levels of circulating factors such as hormones, inflammatory cytokines, and immune cells can significantly alter BBB leakiness.The objective of this study was to discover novel biomarkers of BBB leakiness using the in vitro three‐dimensional Neurovascular Unit (3D NVU; Research Triangle Institute International, Durham, NC) to examine the impact of human blood serum on transcriptional regulation and function of the BBB. The 3D NVU integrates endothelial, neuronal, astrocytic, and microglial cells into a three‐dimensional platform, and allows for the assessment of BBB permeability (transendothelial electrical resistance (TEER, Ω cm2)). Blood serum of secondary progressive MS patients (two men, two women) and four healthy individuals (two men, two women) were purchased from PrecisionMed, Inc. (Solana Beach, CA), and diluted to 40% with cell culture media for testing. TEER of the 3D NVU was recorded immediately prior to serum exposure (baseline), and again at 24 hours after serum treatment. At the end of the experiment, cells of the 3D NVU were collected, and expression of 84 MS‐related genes were measured by quantitative RT‐PCR. BBB permeability was calculated as TEERSerum – TEERBaseline. The protein levels of 100 serum cytokines were measured in undiluted serum samples by membrane‐based sandwich immunoassay. Multiple regression analysis was used to determine the relationship among TEER, serum cytokine levels, and BBB gene expression.We found that no single serum cytokine was a strong predictor of BBB permeability, however, serum‐induced changes in expression of chemokine ligand 12 (Ccl12/Mcp‐5), chemokine ligand 7 (Ccl7), hexosaminidase subunit beta (Hexb), interleukin 13 (Il13), interleukin 1 beta (Il1b), protein tyrosine phosphatase non‐receptor type 11 (Ptpn11), and vascular adhesion molecule 1 (Vcam1) were strong and significant predictors of BBB permeability (R2’s ≥ 0.76, P’s ≤ 0.004). Some of these genes (Il1b, Hexb and Vcam1) have been shown previously to play a role in regulation of BBB function, however, genes such as Ptpn11 have not yet been implicated in BBB permeability. This preliminary study demonstrates that the 3D NVU can be used to elucidate novel, physiologically relevant biomarkers of BBB‐related neurological and psychological disorders. In the future, we plan to perform RNA‐sequencing to examine a broader range of differential gene expression in our study.Support or Funding InformationFunded by TATRC AMEDD Advanced Medical Technology Initiative grant. The views expressed in this abstract are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.