CpG-ODN promotes phagocytosis and autophagy through JNK/P38 signal pathway in Staphylococcus aureus-stimulated macrophage

Abstract

Aims: Phagocytic and autophagic responses are critical for effective host defense against bacterial infection. Bacterial DNA which contains unmethylated Cytosine-phosphate-Guanine (CpG) motifs can trigger a variety of defense mechanisms via Toll-like receptor 9 (TLR9). Here, we aimed to investigate the underlying mechanism of TLR9-mediated phagocytosis and autophagy in Staphylococcus aureus (S.aureus)-stimulated macrophages.

Main methods: The macrophage cell line RAW264.7 or primary peritoneal macrophage was pretreated with CpG-ODN and then stimulated by S. aureus, where some of them were pretreated with SP600125 or SB203580 simultaneously. The protein expressions of TLR9, MyD88, SR-A, CD36, LC3, Beclin-1, and phosphorylated level of c-Jun N-terminal kinase (JNK), P38 and extracellular-regulated protein kinase (ERK) were detected by western blotting. The phagocytosis and LC3 punctate-structures of macrophage were observed by confocal laser scanning microscope.

Key findings: CpG-ODN significantly amplified S. aureus-induced phagocytosis and autophagy of RAW264.7 and TLR9(+/+) primary peritoneal macrophage as compared to that of Non-CpG treated cells, while such effect was abolished in TLR9(-/-) primary peritoneal macrophages. Meanwhile, CpG-ODN significantly enhanced S. aureus-induced phosphorylation of JNK and P38 but not ERK in RAW264.7. Specific inhibition of JNK or P38 by SP600125 or SB203580, dramatically down-regulated CpG-induced phagocytosis and autophagy in S. aureus-stimulated RAW264.7 and TLR9(+/+) primary peritoneal macrophage, while they showed no further down-regulation of phagocytosis and autophagy in TLR9(-/-) primary peritoneal macrophages.

Significance: Our data indicated that CpG-ODN activates TLR9-JNK/P38 signaling to promote phagocytosis and autophagy in S. aureus-stimulated macrophages, these findings provide novel insights into how innate immune cells defend bacterial infection via TLR9.

Keywords: Autophagy; Bacterial defense; CpG; Macrophage; Phagocytosis; Toll-like receptor 9.