Abstract 13967: Macrophages Store Low Density Lipoprotein-Delivered Microbial Small RNAs in Lipid Droplets

Abstract

Low-density lipoproteins (LDL) are causal factors in atherosclerosis. Inflammation is a driving force for both atherogenesis and advanced lesion progression. LDL stimulates inflammation in the lesion; however, the mechanism by which LDL activates macrophages and promotes atherosclerosis inflammation remains to be fully defined. Recently, we reported that LDL is enriched with microbial small RNAs (msRNA) derived from bacteria in the microbiome and environment. Most importantly, we showed that LDL-delivered msRNA activated toll-like receptor 8 (TLR8) in recipient macrophages and inhibition of this pathway reduced atherosclerosis. Nevertheless, the processes by which macrophages store and catabolize exogenous LDL-delivered msRNA are unknown. We hypothesize that LDL-delivered msRNA is stored in lipid droplets of macrophages . LDL was found to transport approximately 37μg of total RNA per mg of protein, and macrophages were found to take up LDL-msRNAs through an LDL receptor-independent process (pinocytosis). Confocal microscopy and RNA quantification of purified organelles indicate that macrophage lipid droplets (LD) contain RNA and that LDL treatment increases LD-associated RNA. Macrophages transfected with msRNA fluorescence-labeled (msRNA-Cy5) contain signal in both the organic and aqueous phases after Bligh-Dyer lipid extraction, indicating a potential acyl esterification of the msRNA. To evaluate LDL-msRNA delivery to macrophages in vitro , cells were treated with reconstituted LDL (rLDL) loaded with msRNA-Cy5. Utilizing confocal microscopy, rLDL was found to deliver msRNA-Cy5 to macrophages. Ongoing research aims to determine the subcellular processing of LDL-delivered msRNA in macrophages; however, results from microscopy studies support that the endo-lysosome and LD compartments are involved. Moreover, rLDL provides a powerful tool for the study of LDL-msRNA delivery to macrophages by reducing the heterogeneity of lipid cargo that may confound analyses, while enabling the enrichment of specified RNA cargo for cellular delivery. These results support that i) LDL can transfer pro-inflammatory msRNA to macrophages, ii) LDL treatment increases LD-associated RNA, and iii) RNA can be isolated by organic lipid extraction.