Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) that affects 400 thousand Americans. In MS attacks, peripheral blood leukocytes gain access to the brain parenchyma and attack white matter structures. Effective therapies for MS target the steps by which these leukocytes gain access to the brain, called the leukocyte adhesion cascade. These therapies, while effective, can cause rare infectious complications and exhibit variability in response between individuals. A more complete understanding of the leukocyte adhesion cascade may lead to therapies that are safer and effectively treat disease in patients that do not respond to current treatment. Crawling of leukocytes within the blood vessels towards sites of inflammation is an important part of the leukocyte adhesion cascade and is the least understood of its steps. Chemokines may guide the directed intravascular crawling of leukocytes, but proof and mechanistic detail are lacking. Most current knowledge about crawling comes from in vivo studies which are limited in scope due to low throughput. An in vitro model to enable the dissection of this process in fine detail and with high throughput would advance the field. To address this, I designed a microfluidic device that uses principles of laminar flow to allow careful studies of the mechanisms underlying directed leukocyte crawling for the first time in vitro. This device allows high resolution live video imaging of human peripheral blood mononuclear cells (PBMC) and their adhesion cascade interactions with human brain microvascular endothelial cells (hBMEC). Production of this device and cell culture within it are now routine, allowing us to pursue the objective of establishing whether endothelial cells guide leukocyte intravascular crawling via chemokines. The project has two specific aims.
Aim 1 will answer whether intraluminal chemokine gradients can plausibly direct intravascular crawling by testing whether an acellular chemokine gradient is sufficient to induce directed crawling of leukocytes. This will be extended by quantifying leukocyte crawling behavior on a directionally-stimulated endothelium in the device. Finally, the contribution of chemokine receptors to these phenomena will be assessed, using drugs to block downstream signaling and antibodies to block ligation.
Aim 2 seeks to shed more light on an alternate pathway that has been proposed: direct signaling of endothelial cells to leukocytes via Golgi-derived vesicles laden with chemokine. To do so, we will use lattice light sheet microscopy (LLSM) to quantify chemokine-laden vesicle trafficking with a high degree of spatiotemporal resolution. We will use this information to test whether the trafficking of these vesicles is influenced by fluid flow, proximity to endothelial-endothelial boundaries, and the presence of adherent leukocytes. The proposed experiments will significantly advance our understanding of leukocyte intravascular crawling, as well as provide refined models for future studies of this phenomenon, which has implications for cell-mediated autoimmune diseases such as MS.
Inflammation controlled by the blood-brain barrier (BBB) plays a major role in multiple sclerosis (MS), and may contribute to the progression of other neurological diseases such as Alzheimer's disease and vascular dementias. Drugs that block inflammation can prevent MS attacks, but also cause rare and severe side effects. This project aims to shed new light on early events in BBB inflammation, hopefully providing new targets for therapies against MS and inflammatory neurodegenerative diseases.
| Benson, Bryan L; Li, Lucy; Myers, Jay T et al. (2018) Biomimetic post-capillary venule expansions for leukocyte adhesion studies. Sci Rep 8:9328 |
| Rathkey, Joseph K; Benson, Bryan L; Chirieleison, Steven M et al. (2017) Live-cell visualization of gasdermin D-driven pyroptotic cell death. J Biol Chem 292:14649-14658 |
