Department of Pediatrics, The First Affiliated Hospital of Anhui Medical University, 218 Jixi Road, Shushan District, Hefei City 230022, Anhui Province, China.
Department of Pediatrics, The First Affiliated Hospital of Anhui Medical University, 218 Jixi Road, Shushan District, Hefei City 230022, Anhui Province, China.
Immunobiology. 2022 May;227(3):152223. doi: 10.1016/j.imbio.2022.152223. Epub 2022 May 4.
The present study intends to clarify the hypothesis that PVL-positive Methicillin-resistant S. aureus strain (PVL-MRSA)-infected macrophages regulate autophagy and thus in turn inhibit phagocytosis through the in vitro and in vivo experiments. The autophagy of mouse macrophage cell line RAW264.7 was observed by fluorescence microscopy, and counted based on the number of each cell dot-like structure GFP-LC3. The protein levels of the phagocytic factors associated with autophagy were determined by western blotting. The phagocytosis of RAW264.7 on MRSA was determined by counting the colony. The clinically isolated and identified PVL-MRSA strain was used to infect BALB/c mice (left nasal drip) to establish a mouse pneumonia model. PVL-MRSA mice were then treated with 3-MA or linezolid. Bronchoalveolar lavage fluid (BALF) from mice was collected for macrophage counting by Flow cytometry assay. The right lung was aseptically isolated for counting the amount of bacteria. The results showed that PVL-MRSA could induced the autophagy of macrophages, which in turn reduced the damage from macrophages, which were respectively alleviated by 3-MA and aggravated by rapamycin. Exogenous rPVL administrated into PVL-MRSA-infected macrophages caused the autophagy of macrophage. Exogenous rPVL, particularly A-Luk S-PV, administrated into macrophages also caused the autophagy of macrophage, which was reversed by PMX53, a C5aR antagonist. In a mouse pneumonia model, PVL-MRSA could induced the autophagy of macrophages, which in turn reduced the damage from macrophages, which were respectively alleviated by 3-MA or linezolid. In conclusion, this study indicated PVL-MRSA regulated macrophage autophagy, which in turns inhibit the phagocytosis of S. aureus by macrophage. This study may provide a potential target against S. aureus infection.
携带 PVL 的耐甲氧西林金黄色葡萄球菌（PVL-MRSA）感染的巨噬细胞可通过调控自噬，进而抑制吞噬作用。通过荧光显微镜观察 RAW264.7 巨噬细胞自噬，根据每个细胞点状结构 GFP-LC3 的数量进行计数。通过 Western blot 测定与自噬相关的吞噬因子的蛋白水平。通过计数 MRSA 对 RAW264.7 的菌落数来确定 RAW264.7 的吞噬作用。采用临床分离鉴定的 PVL-MRSA 株感染 BALB/c 小鼠（左侧滴鼻）建立肺炎模型。然后用 3-MA 或利奈唑胺处理 PVL-MRSA 小鼠。通过流式细胞术检测小鼠支气管肺泡灌洗液（BALF）中的巨噬细胞计数。无菌分离小鼠右肺，计数细菌量。结果表明，PVL-MRSA 可诱导巨噬细胞自噬，进而减轻巨噬细胞的损伤，3-MA 减轻、雷帕霉素加重这种作用。外源性 rPVL 加入 PVL-MRSA 感染的巨噬细胞中，可诱导巨噬细胞自噬。外源性 rPVL，尤其是 A-Luk S-PV，加入巨噬细胞中也可诱导巨噬细胞自噬，C5aR 拮抗剂 PMX53 可逆转这种作用。在肺炎小鼠模型中，PVL-MRSA 可诱导巨噬细胞自噬，进而减轻巨噬细胞的损伤，3-MA 或利奈唑胺分别减轻、加重这种作用。综上所述，本研究表明 PVL-MRSA 调控巨噬细胞自噬，进而抑制巨噬细胞吞噬金黄色葡萄球菌。本研究可能为金黄色葡萄球菌感染提供潜在的治疗靶点。
