Investigating the effect of intrinsic and extrinsic cues of Alzheimer’s disease on immune cell trafficking via in vitro models of the blood-brain barrier

Introduction: Alzheimer’s disease (AD) is the most common neurodegenerative disease, leading to cognitive decline. Dysfunction of the blood-brain barrier (BBB) – a semi-permeable cellular component that separates the central nervous system from the circulatory system – plays a fundamental role in AD progression. Given the complex nature of this disease, experimental models are essential to better understanding AD pathogenesis and informing treatment development. Previously, we have engineered 3D in vitro models of the BBB with intrinsic and extrinsic cues of AD. Intrinsic cues include seeding microfluidic devices with human-induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial cells (iBMECs) containing mutations linked to familial AD (e.g., APP(Swe)). Extrinsic cues, specifically age-related circulatory cues, are incorporated by perfusing microvessels with aged versus young human serum.

In addition to genetic and age-associated factors, research shows that neuroinflammation plays a fundamental role in the pathogenesis of AD. Under normal conditions, the BBB blocks leukocytes from transmigrating into the brain. However, during AD, resident immune cells secrete pro-inflammatory cytokines which activate BMECs. This activation stimulates leukocyte adhesion and transmigration. Leukocyte accumulation in vasculature is associated with impeded flow and cognitive impairment in in vivo AD mouse models. Due to growing evidence concerning neuroinflammation as a driver of AD, we sought to determine the effect of intrinsic and extrinsic cues on immune cell trafficking.

Materials and

Methods: Microfluidic devices were fabricated to create 3D in vitro BBB models using previously published protocols. Devices were either seeded with wild-type (WT) or amyloid precursor protein knock-in (APP(Swe)) iBMECs and perfused with medium containing young or aged human serum. In total, four experimental conditions were tested: (1) WT iBMEC + young serum (control group), (2) APP iBMEC + young serum (intrinsic experimental group), (3) WT iBMEC + aged serum (extrinsic experimental group), and (4) APP iBMEC + aged serum (intrinsic and extrinsic experimental group).

To assess immune cell trafficking, immune cell adhesion assays were performed on day 0 and day 4 following exposure to serum (Figure 1a). Leukocyte adhesion was evaluated by perfusing briefly with fluorescently labeled THP-1 cells, a monocytic cell line, at a concentration of 1 x 10^6 cells per mL. After washing away non-adherent cells, remnant cells were counted manually. Microvessels were then fixed, and cross sections were stained for immune cell adhesion molecules, specifically ICAM-1 and VCAM-1 (Figure 2a). Marker levels were measured by fluorescence intensity.

Results, Conclusions, and Discussions: Results and

Discussion:
Data analysis revealed quantitative differences in immune cell trafficking between control and AD BBB microvessels. Immune cell adhesion experiments demonstrated that intrinsic and extrinsic cues increase the number of adherent THP-1 cells (Figure 1b-c). Compared to day 0, the APP iBMEC + aged serum experimental group exhibited the highest fold-increase in adherent cells (>1 fold-change of adherent THP-1 cells per cm). Conversely, the APP iBMEC + young serum and WT iBMEC + aged serum experimental groups showed a similar fold-change in adherent cells to the WT iBMEC + young serum control. This suggests that when combined, intrinsic and extrinsic cues of AD may have a synergistic effect that facilitates leukocyte aggregation.

Immunostaining experiments indicated that intrinsic and extrinsic cues also upregulate immune cell adhesion molecules (Figure 2b). Relative to the WT iBMEC + young serum control, the APP iBMEC + young serum, WT iBMEC + aged serum, and APP iBMEC + aged serum experimental groups displayed higher normalized fluorescence intensity levels of ICAM-1 and VCAM-1. The highest intensity levels were observed in the APP iBMEC + aged serum experimental group, again suggesting that combined intrinsic and extrinsic cues may synergistically promote immune cell trafficking. Interestingly, VCAM-1 showed a more marked increase than ICAM-1 across all groups. VCAM-1 is predominantly expressed on vascular endothelial cells and facilitates initial leukocyte tethering and rolling. Thus, dysfunctional BBB expression of VCAM-1 may be a key contributor to neuroinflammation in AD.

Conclusion:
To investigate the effect of intrinsic and extrinsic cues of AD on immune cell trafficking, we utilized 3D in vitro models of the BBB. Immune cell adhesion assays highlighted a synergistic increase in leukocyte adhesion with the incorporation of intrinsic mutations and extrinsic aged serum. Immunostaining of adhesion molecules revealed that intrinsic and extrinsic cues also upregulate ICAM-1 and VCAM-1, with VCAM-1 experiencing a more pronounced effect. This study demonstrates the potential role of intrinsic and extrinsic cues in exacerbating neuroinflammation and promoting the pathological progression of AD.

Acknowledgements (Optional): Thank you to Tracy Chung and Dr. Peter Searson for their steadfast guidance and support. Special thanks to the Institute for NanoBioTechnology and the Department of Biomedical Engineering at Johns Hopkins University for the opportunity to learn and conduct this research project.