(P30-040-23) Macrophage Exosomes Transfer miR-3064-5p to Target IκBα and Activate NF-κB Signaling: A Novel Mechanism of Palmitate-induced Metabolic Inflammation

Objectives: Exosomes (EXO) are cell-secreted vesicles with a size of 40~160 nm and play vital roles in intercellular communication. Macrophages (Mø) EXO mediate metabolic inflammation (MI) by transporting microRNAs (miRs) to regulate metabolic organs. Palmitate (PA) is known to cause MI. This study aims to investigate if and how Mø EXO and its miRs mediate the PA-induced MI.

Methods: EXO secreted by RAW264.7 mouse Mø stimulated with PA (PmEXO) or not (mEXO) were prepared by ultracentrifugation. The differential miRs between PmEXO and mEXO were identified by high-throughput sequencing, and their targeted mRNAs and proteins were bioinformatically analyzed and verified by qPCR and western blot. Mouse RAW264.7 Mø, AML-12 hepatocytes, C2C12 myocytes or 3T3-L1 adipocytes were treated with PmExo or mExo. The verified miR and its targeted molecules related to inflammation were analyzed in recipient cells. Further, EXO were prepared from primary peritoneal Mø isolated from AIN93G diet-fed (ImEXO) or 20% PA-contained high fat diet-fed (IPmEXO) mice. ImEXO and IPmEXO were then tail vein injected (30μg) into mice (n=10), respectively twice a week for 2w. The verified miR and its targets in blood, blood EXO, and metabolic tissues were detected.

Results: Both mEXO and PmEXO had an oval double membrane structure with a mean size of ~120.5 nm and rich of EXO-specific marker TSG101 and CD9. PmEXO harbored 81 up-regulated and 56 down-regulated miRs compared to mEXO, among which miR-3064-5p was 5.93 times up-regulated. Bioinformatics revealed that miR-3064-5p targeted to inhibit IκBα in NF-κB pathway. qPCR verified that miR-3064-5p was significantly (p < 0.05) elevated in PmEXO (vs. mEXO). PmEXO treatment significantly (p < 0.05) elevated miR-3064-5p, decreased IκBα, and increased NF-κB p-p65/p65 and cytokines (TNFα, IL-6 and IL-1β) in Mø, hepatocytes, myocytes and adipocytes, whereas mEXO didn’t. Further, IPmEXO (vs. ImEXO) injection significantly (p < 0.05) increased miR-3064-5p in blood, blood EXO, liver, muscle and adipose tissue, and decreased IκBα and increased NF-κB p-p65/p65 and cytokines (TNFα, IL-6 or IL-1β) in liver, muscle and adipose tissue of recipient mice.

Conclusions: Mø Exo transfer miR-3064-5p to target IκBα and activate NF-κB signaling is a novel mechanism of PA-induced MI.

Funding Sources: National Natural Science Foundation of China (NSFC, No. 82073532; 81872621)