Effect of infected bone marrow suspension on gene expression in RAW264.7 macrophages. Effect of infected bone marrow suspension on gene expression in RAW264.7 macrophages

Bacterial infectious diseases have posed a serious challenge to public health, often resulting in treatment failure and infection recurrence due to the emergence of drug-resistant bacteria. Owing to inaccessible binding sites, pathogens can evade attack from host immune cells and traditional antibiotics, leading to local immunosuppressive status. Our study reports a novel bacteriophage-based immune scavenger labeling nanoplatform (Mn2+@Man-phage) to combat immune-evasive bacteria and reverse immunosuppressive status. Our nanosystem utilizes the inherent bacterium-targeting ability of bacteriophages to aggregate at infection sites and mediates mannose-dependent recognition, phagocytosis, and killing of bacteria by macrophages, while the released Mn2+ amplifies the antibacterial immune efficacy. Consequently, macrophages polarize towards M1 and secrete various pro-inflammatory factors, effectively clearing bacteria. Moreover, reprogramming macrophages directly activate T cells at infection sites, eliciting potent adaptive antibacterial immune responses and ultimately achieving bacterial eradication. Overall, we demonstrate a universal strategy for pathogen targeting and immunomodulation of macrophages against bacterial infection. Overall design: To investigate the effect of infected bone marrow suspension on gene expression in RAW264.7 macrophages, the mouse implant-related skeleton infection model was established on the BALB/c mouse tibiae. After 7 days post E.coli infection, the tibiae were harvested and the bone marrow was flushed out with high-glucose medium to obtain bone marrow suspension. Then the bone marrow suspension was filtrated through a 0.22μm filter to culture RAW264.7 cells for 24 h. After co-cultured with E. coli for 6 h, the cells were collected and the total RNA was extracted.

细菌性传染病已对公共卫生构成严峻挑战，耐药菌的出现常导致治疗失败与感染复发。由于病原体难以接触结合位点，可逃避宿主免疫细胞与传统抗生素的攻击，进而引发局部免疫抑制状态。本研究报道了一种新型基于噬菌体（bacteriophage）的免疫清除剂标记纳米平台（Mn²⁺@Man-噬菌体），用于对抗免疫逃逸型细菌并逆转局部免疫抑制状态。该纳米系统利用噬菌体固有的细菌靶向能力在感染部位聚集，并介导甘露糖依赖的巨噬细胞识别、吞噬与杀伤细菌过程，而释放的Mn²⁺可放大抗菌免疫功效。由此，巨噬细胞极化为M1型并分泌多种促炎细胞因子，有效清除细菌。此外，重编程巨噬细胞可直接激活感染部位的T细胞，诱发强效适应性抗菌免疫应答，最终实现细菌的彻底清除。整体实验设计：为探究感染骨髓悬液对RAW264.7巨噬细胞基因表达的影响，本研究在BALB/c小鼠胫骨上构建了小鼠植入物相关骨骼感染模型。大肠杆菌（E.coli）感染7天后，采集胫骨并用高糖培养基冲洗骨髓以获取骨髓悬液。随后将骨髓悬液通过0.22μm滤膜过滤后，与RAW264.7细胞共培养24小时。将细胞与大肠杆菌共培养6小时后，收集细胞并提取总RNA。