Lactobacillus mucosae DPC 6426 as a Bile-Modifying and Immunomodulatory Microbe

DATA Four separate documents are contained herein. The first pertains to bile deconjugation profiling and bile salt hydrolase activity displayed by a number of Lactobacillus mucosae and Lactobacillus reuteri strains. The sheets contained within this file include "Protein Extract BSH Assay", "Bile Deconjugation by Class" and "Bile Deconjugation Complete", with the latter two containing data for both murine and porcine bile exposures. The second document contains cytokine gene expression and media concentrations of same following exposures of RAW264.7 macrophages to varying levels of exopolysaccharide (EPS) and secretome of Lactobacillus mucosae DPC 6426. Data includes IL-10, IL-6, TNF-a, CD206 and iNOS expression, as well as medium concentrations of IL-10 and NO, and cell viability assays. The third contains reactive oxygen species release results from both promyelocyte cell line and human isolated neutrophils following exposures to varying levels of EPS. The final document contains data relating to TLR reporter assays in human embryonic kidney cells following exposures to varying levels of EPS. ABSTRACT Background | Lactobacillus mucosae DPC 6426 has previously demonstrated potentially cardio-protective properties, in the form of dyslipidaemia and hypercholesterolemia correction in an apolipoprotein-E deficient mouse model. This study aims to characterise the manner in which this microbe may modulate host bile pool composition and immune response, in the context of cardiovascular disease. Lactobacillus mucosae DPC 6426 was assessed for bile salt hydrolase activity and specificity. The microbe was compared against several other enteric strains of the same species, as well as a confirmed bile salt hydrolase-active strain, Lactobacillus reuteri APC 2587. Results | Quantitative bile salt hydrolase assays revealed that enzymatic extracts from Lactobacillus reuteri APC 2587 and Lactobacillus mucosae DPC 6426 demonstrate the greatest activity in vitro. Bile acid profiling of porcine and murine bile following incubation with Lactobacillus mucosae DPC 6426 confirmed a preference for hydrolysis of glyco-conjugated bile acids. In addition, the purified exopolysaccharide and secretome of Lactobacillus mucosae DPC 6426 were investigated for immunomodulatory capabilities using RAW264.7 macrophages. Gene expression data revealed that both fractions stimulated increases in interleukin-6 and interleukin-10 gene transcription in the murine macrophages, while the entire secretome was necessary to increase CD206 transcription. Moreover, the EPS elicited a dose-dependent increase in nitric oxide and interleukin-10 production from RAW264.7 macrophages, concurrent with increased tumour necrosis factor- secretion at all doses. Conclusions | This study indicates that Lactobacillus mucosae DPC 6426 modulates both bile pool composition and immune system tone in a manner which may contribute significantly to the previously identified cardio-protective phenotype.本数据集包含四份独立文档。 第一份文档聚焦黏膜乳杆菌（Lactobacillus mucosae）与罗伊乳杆菌（Lactobacillus reuteri）多个菌株所展现的胆汁去结合谱与胆汁盐水解酶（bile salt hydrolase, BSH）活性。该文件内的工作表包括「蛋白质提取物BSH测定」「按类别划分的胆汁去结合情况」以及「完整胆汁去结合数据」，后两者包含了小鼠与猪胆汁暴露实验的数据。 第二份文档包含RAW264.7巨噬细胞暴露于不同浓度的黏膜乳杆菌DPC 6426的胞外多糖（exopolysaccharide, EPS）与分泌组后，细胞因子基因表达量及对应培养基浓度数据。检测指标包括IL-10、IL-6、TNF-α、CD206与iNOS的基因表达，同时涵盖培养基中IL-10与NO的浓度，以及细胞活力测定结果。 第三份文档包含早幼粒细胞系与人类分离中性粒细胞暴露于不同浓度EPS后的活性氧释放实验结果。 第四份文档包含人类胚胎肾细胞暴露于不同浓度EPS后的Toll样受体（Toll-like receptor, TLR）报告基因测定相关数据。 摘要 背景 | 此前研究显示，黏膜乳杆菌DPC 6426可通过改善载脂蛋白E缺陷小鼠模型的血脂异常与高胆固醇血症，展现出潜在的心血管保护特性。本研究旨在明确该微生物在心血管疾病背景下，调控宿主胆汁池组成与免疫应答的具体方式。我们对黏膜乳杆菌DPC 6426的胆汁盐水解酶活性与特异性进行了评估，并将其与该菌种的其他数株肠道菌株，以及一株确认具有胆汁盐水解酶活性的罗伊乳杆菌APC 2587进行了对比。 结果 | 定量胆汁盐水解酶测定结果显示，罗伊乳杆菌APC 2587与黏膜乳杆菌DPC 6426的酶提取物在体外展现出最高的酶活性。将黏膜乳杆菌DPC 6426与猪、小鼠胆汁共孵育后的胆汁酸谱分析证实，该菌株偏好水解甘氨酸结合型胆汁酸。此外，我们利用RAW264.7巨噬细胞探究了黏膜乳杆菌DPC 6426的纯化胞外多糖与分泌组的免疫调节能力。基因表达数据显示，两种组分均可促进小鼠巨噬细胞中白细胞介素-6与白细胞介素-10的基因转录，而完整的分泌组才能够上调CD206的转录水平。进一步研究发现，EPS可剂量依赖性地促进RAW264.7巨噬细胞产生一氧化氮与白细胞介素-10，同时在所有测试剂量下均能增加肿瘤坏死因子-α的分泌。 结论 | 本研究表明，黏膜乳杆菌DPC 6426可通过调控胆汁池组成与免疫系统状态，为此前已发现的心血管保护表型提供重要的潜在机制。