Macrophage-Derived Exosomes in TLR9-/- Mice Ameliorate Sepsis-Induced Mitochondrial Oxidative Stress and Apoptosis in Cardiomyocytes.

Abstract

Objective To explore the mechanisms of TLR9 from macrophages on mitochondrial apoptosis in cardiomyocytes at early stage of sepsis. Methods The in vivo and in vitro sepsis mice were bone marrow transplantation (BMT) with wild type (WT) or (toll-like receptor 9) TLR9 knockout (−/− or KO) myeloid cells and then constructed by cecum ligation and puncture (CLP) as vivo experiment and cardiomyocytes cocultured with WT or TLR9-deficient macrophages treated with LPS as vitro experiment, respectively. Sepsis model were performed by CLP. The expression levels of exosome, PI3K/AKT, and ERK1/2, inflammatory factors, and apoptotic proteins were tested by western blot in vivo. Besides, associated apoptotic proteins and JC-1 fluorescence assay were tested in vitro. Results The expressions of p-PI3K, p-AKT, exosome markers (CD9, CD63, and TSG101), p-ERK1/2, TNF-α, IFN-γ, IL-1β, and cleaved-caspase-3/-9 were significantly increased in septic mice vs. control mice, and these proteins were declined dramatically in TLR9−/− BMT mice vs. WT BMT mice in sepsis mice models. Meanwhile, the protein expression of cytochrome C, cleaved-caspase-3, and cleaved-caspase-9 increased significantly in primary mouse myocardial cells cocultured with TLR9−/− or WT macrophages stimulated with LPS, and these mitochondrial apoptotic proteins as well as the green 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) fluorescence were dramatically lower in LPS-stimulated cardiomyocytes cocultured with TLR9−/− than with WT macrophages. Conclusion TLR9−/− in macrophages suppressed the inflammatory reaction as well as the exosome secretion and resulted in the inhibition of apoptosis and oxidative stress in sepsis-induced cardiomyopathy.