(683f) Adipocyte Extracellular Vesicles (AdEVs) Uptake and Effects on Leukocyte Functionality: Obese Vs. Lean

Obesity is an ever-rising trend with consequences on health, such as increased inflammation. A unique activity amongst leukocytes is displayed in obese patients. To understand the ties between obesity and inflammation there must be an identification of the bio-communication between adipocytes and immune cells. In obesity, adipocytes (Ad) are known to produce an increased number of extracellular vesicles (EVs), as EVs contain important bio-communication components such as microRNA, RNA, and lipids. Therefore, this study utilized EVs from adipocytes collected from obese and lean individuals, then added to human peripheral blood neutrophils and monocytes to understand the impacts of obese AdEVs on immune cell dynamics and function.

Methods

Adipose tissue is collected during bariatric surgery, and isolated adipocytes are cultured to allow for the generation of AdEVs. Culture media is purified through tangential flow filtration, and the EV concentration is measured through tunable resistive pulse sensing. Human blood is drawn from a lean healthy donor control, and human peripheral blood neutrophils and monocytes are isolated through negative immunoselection. RNA cargo within the EVs is hybridized to a molecular beacon targeting miR-21-5p to allow for visualization of uptake. AdEVs are allowed to incubate with human neutrophils, at a concentration of 10,000 EVs per cell, and added to an engineered bio-particle microarray with clusters of heat-killed bacteria. The wide-scale array is designed with uniform diameter and spacing to mimic neutrophil's pathogenic recognition. This array allows for the high-throughput investigation of swarming neutrophil dynamics in vitro. AdEVs were added to monocytes in the same manner.

Results / Discussion

Fluorescent signal of miR-21-5p-EV uptake by neutrophils was quantified through confocal microscopy. Obese AdEVs significantly impact neutrophil swarming dynamics and ability to undergo coordinated migration, where there are significantly increased number of cells outside of the neutrophil swarm when they are influenced by obese AdEVs (Figure 1A), neutrophil migratory tracks significantly decrease in linearity compared to lean AdEVs, non-activated neutrophil EVs (NaNEVs), and native neutrophils (Figure 1B), and the area of the neutrophil swarm is significantly less after 1 hour for obese AdEVs compared to lean AdEVs, NaNEVs, and native neutrophils (Figure 1C). AdEVs produced morphological changes in human peripheral blood monocytes and increased proinflammatory markers measured by qRT-PCR.

Conclusions

A method was generated for EV-RNA uptake visualization that does not require a micelle-forming lipophilic dye. AdEVs impact immune cells in multiple facets, causing neutrophils to have a reduced ability to undergo coordinated migration when presented heat-killed bacteria or both neutrophils and THP-1 cells to be hyperactive in their natural state. The implications of this study help identify the ties between the adipocytes and the immune system through their means of bio-communication, EVs.