Hui-Mei Wu1, Jiong Wang1, Bing Zhang1, Lei Fang1, Ke Xu1, Rong-Yu Liu2. 1. Anhui Geriatric Institute, The First Affiliated Hospital of Anhui Medical University, Jixi Road 218, Hefei, Anhui 230022, PR China. 2. Anhui Geriatric Institute, The First Affiliated Hospital of Anhui Medical University, Jixi Road 218, Hefei, Anhui 230022, PR China. Electronic address: rongyuliu@hotmail.com.
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.
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.
Authors: Eleonora Ciarlo; Tytti Heinonen; Charlotte Théroude; Jacobus Herderschee; Matteo Mombelli; Jérôme Lugrin; Marc Pfefferlé; Beatrice Tyrrell; Sarah Lensch; Hans Acha-Orbea; Didier Le Roy; Johan Auwerx; Thierry Roger Journal: Front Immunol Date: 2017-08-28 Impact factor: 7.561