Supplementary Material for: Augmentation of Urinary Lactoferrin Enhances Host Innate Immune Clearance of Uropathogenic Escherichia coli

Urinary tract infection (UTI) is a prominent global health care burden. Although UTI is readily treated with antibiotics in healthy adults, complicated cases in immune-compromised individuals and the emerging antibiotic resistance of several uropathogens have accelerated the need for new treatment strategies. Here, we surveyed the composition of urinary exosomes in a mouse model of uropathgenic Escherichia coli (UPEC) UTI to identify specific urinary tract defense constituents for therapeutic development. We found an enrichment of the iron-binding glycoprotein lactoferrin in the urinary exosomes of infected mice. In subsequent in vitro studies, we identified human bladder epithelial cells as a source of lactoferrin during UPEC infection. We further established that exogenous treatment with human lactoferrin (hLf) reduces UPEC epithelial adherence and enhances neutrophil antimicrobial functions including bacterial killing and extracellular trap production. Notably, a single intravesicular dose of hLf drastically reduced bladder bacterial burden and neutrophil infiltration in our murine UTI model. We propose that lactoferrin is an important modulator of innate immune responses in the urinary tract and has potential application in novel therapeutic design for UTI.尿路感染（Urinary tract infection，UTI）是全球重大医疗卫生负担。尽管健康成人罹患UTI后可通过抗生素实现有效治愈，但免疫功能低下人群的复杂感染病例，以及多种尿路致病菌日益凸显的抗生素耐药性，进一步催生了新型治疗策略的研发需求。本研究借助尿路致病性大肠杆菌（Uropathogenic Escherichia coli，UPEC）尿路感染小鼠模型，解析尿液外泌体的组成特征，以期筛选可用于治疗开发的特异性尿路防御组分。研究发现，感染小鼠的尿液外泌体中，铁结合糖蛋白乳铁蛋白（lactoferrin）的富集水平显著升高。后续体外实验证实，在UPEC感染过程中，人膀胱上皮细胞可作为乳铁蛋白的分泌来源。本研究进一步验证，外源性给予人乳铁蛋白（human lactoferrin，hLf）可降低UPEC对上皮细胞的黏附能力，并增强中性粒细胞的抗菌功能，包括细菌杀伤与胞外陷阱形成。值得注意的是，在本研究的小鼠UTI模型中，单次膀胱内给药hLf即可显著降低膀胱内的细菌载量，并减少中性粒细胞浸润。本研究提出，乳铁蛋白是尿路先天免疫应答的重要调控因子，有望应用于UTI的新型治疗方案开发。