Abstract 514: High-density Lipoproteins Mediate Bidirectional Flux Of Extracellular Small RNAs In Macrophages

Abstract

HDL are a class of dynamic particles that play critical roles in lipid metabolism, inflammation, and cell signaling. HDL are small, highly-mobile carriers of diverse molecular cargo, that include many types of host and non-host small RNAs (sRNA), as observed by sequencing. We have previously reported that HDL can accept host sRNAs from macrophages and transfer functional miRNAs to recipient endothelial cells; however, HDL’s ability to transfer sRNAs, host or non-host, to macrophages or mediate intercellular communication between immune cells are unknown. Based on preliminary results, we hypothesize that HDL mediates bidirectional flux of extracellular sRNA between macrophages. To demonstrate that HDL accepts sRNAs from macrophages and determine if macrophages have the capacity to export non-host sRNAs to HDL in vitro , macrophages were loaded with a synthetic microbial sRNA (ssRNA40-Cy5) and allowed to efflux to accepting HDL in the media. Macrophage HDL-sRNA acceptance was assessed by size-exclusion chromatography and fluorometry; and macrophages were found to export microbial sRNAs to HDL in a process inhibited by monensin, an ionophore and small molecule inhibitor of the endo-lysosomal pathway. Previous studies of HDL-sRNA flux required targeted candidate approaches based on prior knowledge. To overcome this barrier, sRNAs on intact native HDL particles were labeled (stained) with SYTO RNASelect dye and incubated with macrophages. This approach facilitated the quantification of all sRNAs on HDL using a standard curve and traced sRNA uptake and storage in recipient macrophages. After confirming specificity, SYTO quantification of total sRNAs showed that human HDL transport >14 μg sRNA/ mg total protein, a value far greater than previously recognized. HDL was found to transfer sRNAs to recipient macrophages based on fluorescence quantification and confocal microscopy live-cell imaging of HDL-delivered sRNAs in macrophage organelles. These results support that HDL mediates the bidirectional import and export of extracellular sRNAs from macrophages.