Data Sheet 1_Solithromycin mitigates Prevotella intermedia–induced methicillin-resistant Staphylococcus aureus ventilator-associated pneumonia by enhancing alveolar macrophage function.xlsx

BackgroundVentilator-associated pneumonia (VAP) is a fatal intensive care infection. VAP caused by methicillin-resistant Staphylococcus aureus (MRSA) can be exacerbated by Prevotella intermedia culture supernatant (P. int. sup.). Solithromycin (SOL), a fourth-generation macrolide, inhibits bacterial protein synthesis and modulates immunity; however, its effects on exacerbation of MRSA-VAP by P. int. sup. remain unclear. This study examined whether SOL inhibits bacterial protein synthesis by binding to the 50S ribosomal subunits in P. int. sup. and subsequently reduces the worsening of MRSA-VAP caused by P. int. sup.
MethodsBALB/cCrSlc mice received MRSA and P. int. sup. with or without sub-minimum inhibitory concentrations of SOL (P. int. sup. (SOL)) or clarithromycin (CAM; P. int. sup. (CAM)). Outcomes included survival rates, lung MRSA burden, and transcriptomics (reverse transcription polymerase chain reaction, bulk RNA sequencing [RNA-seq]). In vitro, bone marrow-derived alveolar macrophage-like cells (AMLCs) from C57BL/6J mice were infected with MRSA ± SOL; bactericidal activity and mRNA expression were measured.
ResultsP. int. sup. increased mortality, bacterial load, and neutrophilic infiltration; however, P. int. sup. (SOL) significantly improved survival rate (100%, n = 8, ****P < 0.0001), reduced MRSA burden (n = 10–11, **P < 0.01), and enhanced macrophage recruitment (n = 7–8, ****P < 0.001). P. int. sup. downregulated Ccr2 expression (n = 7–8, ***P < 0.001). RNA-seq analysis revealed P. int. sup. (SOL) upregulated macrophage phagocytosis and bactericidal pathways. SOL-pretreated AMLCs infected with MRSA exhibited reduced bacterial burden (n = 8, *P < 0.05 vs control, **P < 0.01 vs CAM-pretreated AMLCs) and upregulated Tnf-α expression (n = 7–8, *P < 0.05 vs control).
ConclusionSOL protects by activating alveolar macrophages and promoting TNF-related responses, suggesting a novel immunomodulatory role for SOL in host defense against exacerbation of MRSA-VAP by P. int. sup.

背景：呼吸机相关性肺炎（Ventilator-associated pneumonia, VAP）是一种致死性重症监护感染。由耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus, MRSA）引发的VAP，可被中间普雷沃菌培养上清液（Prevotella intermedia culture supernatant, P. int. sup.）加重病情。索利霉素（Solithromycin, SOL）作为第四代大环内酯类药物，可抑制细菌蛋白质合成并调节免疫功能；但其对P. int. sup.加重MRSA-VAP的作用机制尚未明确。本研究旨在探讨SOL是否可通过结合P. int. sup.中的50S核糖体亚基抑制细菌蛋白质合成，进而减轻P. int. sup.诱导的MRSA-VAP恶化。
方法：BALB/cCrSlc小鼠被分为四组：仅接受MRSA联合P. int. sup.处理组，以及分别联合亚抑菌浓度索利霉素（P. int. sup.(SOL)组）或克拉霉素（clarithromycin, CAM；P. int. sup.(CAM)组）的处理组。观测指标包括小鼠生存率、肺组织MRSA载量及转录组学分析（逆转录聚合酶链反应、高通量RNA测序[RNA-seq]）。体外实验中，以C57BL/6J小鼠的骨髓源性肺泡巨噬细胞样细胞（bone marrow-derived alveolar macrophage-like cells, AMLCs）为模型，感染MRSA并联合或不联合SOL，检测其杀菌活性与mRNA表达水平。
结果：P. int. sup.可升高小鼠死亡率、细菌载量并促进中性粒细胞浸润；而P. int. sup.(SOL)组可显著提升小鼠生存率（100%，n=8，****P<0.0001）、降低肺组织MRSA载量（n=10~11，**P<0.01）并增强巨噬细胞招募（n=7~8，****P<0.001）。P. int. sup.可下调Ccr2基因表达（n=7~8，***P<0.001）。RNA-seq分析显示，P. int. sup.(SOL)组可上调巨噬细胞吞噬与杀菌相关通路。经SOL预处理后感染MRSA的AMLCs，其细菌载量显著降低（n=8，*P<0.05 vs 对照组，**P<0.01 vs CAM预处理组），且肿瘤坏死因子α（Tumor necrosis factor-α, Tnf-α）的表达水平上调（n=7~8，*P<0.05 vs 对照组）。
结论：SOL可通过激活肺泡巨噬细胞、促进TNF相关免疫应答发挥保护作用，提示其在宿主防御P. int. sup.加重的MRSA-VAP过程中具有全新的免疫调节功能。