Abstract Body (Do not enter title and authors here): Background: Myocardial ischemia/reperfusion (I/R) injury is mainly ascribed to excessive sterile inflammation resulting from the inefficient clearance of dead cardiomyocytes by macrophages (termed efferocytosis). Bacterium-released extracellular vesicles (bEVs) have been shown as a critical mediator in macrophage (MΦ) function and modulating inflammation. However, their possible role in MΦ efferocytosis during I/R has not been investigated.

Methods: BEVs were isolated from the culture supernatants of three probiotic strains include Lactobacillus rhamnosus GG (LGG), Bifidobacterium BB-12, and Escherichia coli Nissle 1917 (EcN). The collected EVs were then added to bone marrow–derived MΦs (BMDMs), followed by incubation with Deep Red (APC)-labelled dead H9c2 cells for determining MΦ efferocytosis, assessed by flow cytometry. For the in vivo experiments, EVs were administered via tail vein injection into cardiac I/R-operated mCherry-transgenic mice. Subsequently, cardiac MΦ efferocytosis and cardiac remodeling will be analyzed. The mechanism underlying bEV-mediated efferocytosis will be assessed by RNA-sequencing and bioinformatics assays.

Results: We observed that LGG-EVs were the best among these three bEVs in stimulating MΦs to engulf dead H9c2 cells. Using mCherry-Tg mice to undergo the ligation of left anterior descending artery for 45 min, LGG-bEVs (2 µg/g) were injected into mice via the tail vein just prior to reperfusion. One day 4 post-I/R, LGG-bEVs-treated mice exhibited a higher capacity of cardiac MΦ efferocytosis, lower levels of cardiac cells death, inflammatory cytokines (IL-6, TNF-α, MCP-1), and inflammatory cell infiltration, compared to PBS-treated I/R mice (n = 6, p < 0.05). Accordingly, at 1-month post-I/R, LGG-bEV-treated mice showed a marked improvement in cardiac function, along with reduced cardiac fibrosis (n = 8, p < 0.05). Mechanistically, RNA-seq and bioinformatic assays identified that LGG-bEVs contained higher levels of 5sRNA-derived short-RNA fragments, which interacted with coding regions of Ddx5, Ywhaz, and Frmd4a genes, three genes known to promote MΦ efferocytosis. Treatment of MΦs with LGG-bEVs greatly upregulated the expression of Ddx5, Ywhaz, and Frmd4a, which was further validated by co-transfection of these gene-expression plasmids with this short-RNA fragment.

Conclusions: This study suggests that LGG-EVs have therapeutic effects against I/R-induced cardiac injury through promoting MΦ efferocytosis.