CFTR mutation abrogates Prevotella regulation of epithelial cell defense against Staphylococcus aureus

Background: The oral anaerobe Prevotella melaninogenica is enriched in the lungs of people with cystic fibrosis (pwCF), yet its functional impact on respiratory tract homeostasis remains incompletely understood. Prior studies identified immune modulatory effects following lung exposure to Prevotella, but the relevance of these findings for CF infections is unknown. Methods: The impact of P. melaninogenica on infection with the CF pathogen Staphylococcus aureus was evaluated using a mouse lung infection model and human respiratory tract cystic fibrosis transmembrane conductance regulator (CFTR) mutant and isogenic wild-type (WT)-corrected CFBE41o- epithelial cells. RNA-sequencing was performed to compare Prevotella-induced signaling programs in WT-corrected versus CFTR mutant cells. Results: P. melaninogenica significantly reduced S. aureus lung infection, which was associated with elevated S. aureus killing by lung neutrophils and impaired S. aureus adherence to epithelial cells. Live or heat-killed Prevotella were sufficient to mediate these effects, which were dependent on the toll-like receptor TLR2. Prevotella impairment of S. aureus adherence also required CFTR function, as this effect was lost in CFTR mutant cells but restored by CFTR modulator therapy. RNA-sequencing identified several antibacterial defense pathways selectively upregulated by Prevotella in WT corrected epithelial cells, correlating with higher IL-8 and IL-6 cytokine production. Conclusions: P. melaninogenica enhanced neutrophil and epithelial defense against S. aureus, but these benefits were lost with CFTR dysfunction. CFTR modulator therapy rescued Prevotella responsiveness in respiratory epithelial cells, highlighting the potential for synergistic effects of host-microbiome interactions and CFTR targeted therapies. Overall design: CFBE41o- CFTR mutant and isogenic WT corrected CFBE41o- epithelial cells in submerged culture were either untreated or treated with heat-killed Prevotella melaninogenica for 24 hours. The experiment is designed to capture the CFTR-dependent gene expression changes in unstimulated and Prevotella stimulated cells.

背景：口腔厌氧菌产黑色素普雷沃菌（Prevotella melaninogenica）在囊性纤维化患者（people with cystic fibrosis，简称pwCF）的肺部中富集，但其对呼吸道稳态的功能性影响仍未完全阐明。既往研究揭示了肺部暴露于普雷沃菌后的免疫调节效应，但上述发现与囊性纤维化感染的相关性尚不明确。
方法：本研究通过小鼠肺部感染模型以及人呼吸道囊性纤维化跨膜传导调节因子（CFTR）突变株与同基因野生型（WT）校正株CFBE41o-上皮细胞，评估产黑色素普雷沃菌对囊性纤维化致病菌金黄色葡萄球菌（Staphylococcus aureus）感染的影响。通过RNA测序（RNA-sequencing），比较野生型校正株与CFTR突变株细胞中普雷沃菌诱导的信号通路变化。
结果：产黑色素普雷沃菌可显著减轻金黄色葡萄球菌肺部感染，该效应与肺部中性粒细胞对金黄色葡萄球菌的杀伤能力增强，以及金黄色葡萄球菌对上皮细胞的黏附能力受损相关。活菌体或热灭活普雷沃菌均可介导上述效应，且该效应依赖于Toll样受体2（TLR2）。普雷沃菌对金黄色葡萄球菌黏附的抑制作用同样需要CFTR功能，该效应在CFTR突变株细胞中消失，但可通过CFTR调节剂治疗恢复。RNA测序结果显示，普雷沃菌可在野生型校正株上皮细胞中选择性上调多条抗菌防御通路，该现象与IL-8及IL-6细胞因子的表达水平升高相关。
结论：产黑色素普雷沃菌可增强中性粒细胞与上皮细胞对金黄色葡萄球菌的防御能力，但该保护效应在CFTR功能异常时丧失。CFTR调节剂治疗可恢复呼吸道上皮细胞对普雷沃菌的应答反应，凸显了宿主-微生物组互作与CFTR靶向治疗协同发挥效应的潜在可能。
整体实验设计：将贴壁培养的CFBE41o- CFTR突变株与同基因野生型校正株CFBE41o-上皮细胞分为两组，分别不予处理，或用热灭活产黑色素普雷沃菌处理24小时。本实验旨在捕捉未刺激与普雷沃菌刺激的细胞中CFTR依赖性的基因表达变化。