Poster BB5 - Bacterial Extracellular Vesicles Destabilize a Human Stem Cell-Derived Blood-Brain Barrier Model

Introduction: Bacterial infections can result in sepsis, a dysregulated host inflammatory response which is the leading cause of death in hospitals. The blood-brain barrier (BBB) is among the tissues that can be destabilized by sepsis, leading to increased vascular permeability and an influx of undesirable cells and molecules that may alter brain chemistry. Sepsis survivors continue to experience reduced BBB integrity following recovery and consequently have a heightened risk of developing neurological disorders.
Bacteria produce numerous proinflammatory factors that can initiate the signaling cascade that results in sepsis. One understudied factor is bacterial extracellular vesicles (bEVs). These nanoscale vesicles harbor various bacterial markers including the well-known inflammatory stimulant lipopolysaccharide (LPS), and may be relatively stable in blood due to their lipid membrane-bound structures. This means that bEVs may persist in the circulation longer than smaller bacterial factors and could play an important role in inflammatory activation of human cells.
In order to study how bEVs interact with the BBB, we developed a lab-on-chip model featuring human induced pluripotent stem cell-derived brain microvascular endothelial cells (BMECs) and pericytes. This allowed us to examine endothelial cell inflammatory activation as well as changes in barrier properties after exposure to LPS and bEVs.

Materials and

Methods: bEVs were isolated from E. coli-conditioned LB Broth via ultracentrifugation and were analyzed by nanoparticle tracking analysis. Human cell studies were conducted in multiwell plates and in microfluidic devices based around an ultrathin nanoporous membrane made from silicon nitride. After 6 days of culture, BMECs were treated with LPS (500 or 10,0000 pg/ml) or bEVs (1x107 or 1x108 particles/ml) for 16 - 18 h. BMEC inflammatory activation was assessed by live-labeling the cells with an anti-ICAM-1 antibody for 15 min prior to fixation with 4% formaldehyde and subsequent incubation with a fluorescent secondary antibody. The ICAM-1 fluorescent signal obtained from microscopy images was averaged and normalized to the number of counterstained nuclei detected in each image. The integrity of endothelial tight junction proteins was investigated by fixing the cells with ice-cold methanol for 20 s followed by labeling with an anti-claudin-5 antibody for 1 h. We examined barrier integrity of cells seeded in the microfluidic devices by introducing lucifer yellow dye to the top side of the nanoporous membrane and collecting solution from below the membrane after incubating 1 h. Fluorescence intensity of the sample was used to determine the concentration of dye that passed through the membrane during the incubation period. To study the impact of cell co-cultures, BMECs were seeded above the nanoporous membrane and pericytes were seeded on the underside of the membranes.

Results, Conclusions, and Discussions: ICAM-1 is known to become upregulated on the surface of endothelial cells during inflammation. bEVs were able to increase ICAM-1 expression even at concentrations as low as 1x107 particles/ml (Fig. 1). It is worth noting that the true concentration of bEVs in the blood of sepsis patients is difficult to determine due to the challenge of separating human extracellular vesicles and bEVs. BMEC exposure to 10,000 pg/ml LPS led to a significant increase in expression of ICAM-1, while 500 pg/ml LPS caused a non-significant change in expression. This was unexpected because 500 pg/ml is near the upper end of the physiological range for LPS concentrations that have been measured in the blood of sepsis patients. If BMEC activation requires a supraphysiological concentration of LPS, this indicates that LPS alone may not play a substantial role in direct proinflammatory stimulation of brain endothelium during sepsis. In the microfluidic device experiments, LPS treatment led to decreases in BMEC expression of tight junction protein claudin-5 as well as increased permeability to lucifer yellow dye. This indicated that the barrier functions of the cells were compromised. The inclusion of pericytes in the co-cultures failed to demonstrate a protective effect against LPS.
We have obtained some of the first results demonstrating that bEVs cause inflammatory activation of iPSC-derived BMEC-like cells. These results were comparable to the effects that we observed with the more commonly-studied LPS. In the future, we plan to determine which bEV components are primarily responsible for initiating proinflammatory signaling. We believe that this knowledge will deepen our understanding of the progression of sepsis and could potentially be applied in the development of anti-inflammatory clinical treatments that could protect the BBB and improve patient outcomes.

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