(D-158) Adipocyte Extracellular Vesicle Uptake via Leukocytes and their Impact on the Immune Response

Introduction:: Obesity is an ever-rising trend, and with that there are consequences on health, such as increased inflammation. A unique activity amongst leukocytes has been shown in obese patients. This study utilized extracellular vesicles (EVs) from adipocytes (Ad) that have been collected from obese individuals and added to human neutrophils and a monocyte-derived cell line (THP-1) to understand the impact of obese adipocyte EVs on immune cell dynamics.


Materials and Methods:: Subcutaneous adipose tissue was collected during bariatric surgery, and the subcutaneous adipocytes (SAd) were isolated and cultured to allow for the generation of SAd EVs. The culture media was purified through tangential flow filtration, and the EV concentration was measured through tunable resistive pulse sensing. Human blood was drawn from a healthy donor the morning of an experiment and neutrophils were isolated through negative immunoselection. RNA cargo within the EVs was hybridized to a molecular beacon targeting miR-21-5p, a commonly expressed miRNA within EVs. SAd EVs were allowed to incubate with human neutrophils, at a concentration of 1000 EVs per cell, and added to a bio-particle microarray with clusters of bacteria. The array was designed with uniform diameter and spacing to simulate neutrophils pathogenic recognition. SAd EVs were added to THP-1 cells in the same manner. This array allows for the investigation of swarming neutrophil dynamics, in vitro.


Results, Conclusions, and Discussions:: Results / Discussion

Molecular beacons were utilized to visualize vesicular miR-21-5p uptake without the shortcomings of micelle formation in lipophilic dyes. SAd EV uptake was visualized during neutrophil swarming (Figure 1A), where EVs with marked cargo showing an increased fluorescence intensity over time versus EVs with no marked cargo, additionally Ad EVs have shown to impact neutrophil swarming dynamics, where the number of cells significantly decreased per swarm (Figure 1B). SAd EVs produced morphological changes in THP-1 cells (Figure 1C) and an increase in proinflammatory markers measured by qRT-PCR. These results imply obese Ad EVs are negatively influencing leukocytes that are present in the tissue and in circulation.

Conclusions

A method was generated for EV-RNA uptake visualization that does not require a lipophilic dye. SAd EVs impact immune cells in multiple facets causing them to react differently when presented a microorganism or be hyperactive in their natural state. This study heightened the knowledge present on how obesity impacts the cellular immune response.


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