Macrophages present a spectrum of phenotypes that mediate both the pathogenesis and resolution of atherosclerotic lesions. Inflammatory macrophage phenotypes are pro-atherogenic, but the natural factors that instigate this polarization are largely unknown. Here, we demonstrate that microbial small RNAs (msRNA) are enriched on LDL and drive pro-inflammatory macrophage polarization and cytokine secretion via activation of the ribonucleic acid sensor toll-like receptor 8 (TLR8). Removal of msRNA cargo during LDL re-constitution yields particles that readily promote sterol loading but fail to stimulate inflammatory activation. Competitive antagonism of TLR8 with non-targeting locked nucleic acids (nt-LNA) was found to prevent nLDL-induced macrophage polarization in vitro, and re-organize lesion macrophage phenotypes in vivo, as determined by single-cell RNA sequencing. Critically, this was associated with reduced disease burden in distinct mouse models of atherosclerosis. These results identify LDL-msRNA as instigators of atherosclerosis-associated inflammation and support alternative functions of LDL beyond cholesterol transport.
Small RNAs (sRNA) are exchanged between cells and tissues by various lipid and protein carriers. Extracellular sRNAs regulate gene expression in recipient cells, including activation of pattern recognition receptors and inflammatory signaling pathways. Based on size-exclusion chromatography (SEC) fractionation of human plasma, a large proportion of extracellular sRNAs are detected in small lipid-associated ribonucleoproteins (50-150 kDa), a class of carriers that have been largely overlooked. Sequential fractionation approaches, mass spectrometry, enzymatic activity assays, and western blotting all support that lipid-associated human plasminogen (PLG) co-purifies with sRNAs in plasma. PLG transports approximately 10 μg total RNA/ mg protein in plasma. Depletion of PLG from plasma by lysine-sepharose resin was also found to reduce plasma miRNA levels. Based on microscale thermophoresis assays, PLG was determined to have moderate binding to single-stranded sRNAs (K d =1.8μM). Immune cells may be a source of sRNAs, as macrophages were found to export both host and non-host sRNAs to PLG in vitro . To identify PLG-associated sRNAs, sRNA sequencing was completed, and results showed that PLG host sRNA are predominantly derived from parent rRNAs (rDR), tRNAs (tDR), yRNA (yDRs), and microRNAs (miRNA). Furthermore, we found that host sRNAs on PLG are significantly altered in subjects with familial hypercholesterolemia (hetFH), including 22 miRNAs, 5 tDRs, and 10 yDRs. PLG was also found to transport many non-host microbial sRNAs derived from bacteria, fungi, and viruses in the microbiome and environment; and genome counts for many of these microbial species were significantly altered on PLG isolated from hetFH subjects. PLG was found to cause dramatic gene (mRNA) expression changes in treated-macrophages; however, it is unknown what fraction of these changes are associated with PLG-sRNA delivery. Collectively, results suggest that PLG transports high levels of extracellular sRNAs from host and non-host species that are sensitive to hypercholesterolemia and disease associated changes. Although the impact of PLG-sRNAs remains to be determined, they hold great potential as therapeutic targets for inflammation.
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.
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