Table_1_Optimal LC-MS metabolomic profiling reveals emergent changes to monocyte metabolism in response to lipopolysaccharide.xlsx

IntroductionImmunometabolism examines the links between immune cell function and metabolism. Dysregulation of immune cell metabolism is now an established feature of innate immune cell activation. Advances in liquid chromatography mass spectrometry (LC-MS) technologies have allowed discovery of unique insights into cellular metabolomics. Here we have studied and compared different sample preparation techniques and data normalisation methods described in the literature when applied to metabolomic profiling of human monocytes.MethodsPrimary monocytes stimulated with lipopolysaccharide (LPS) for four hours was used as a study model. Monocytes (n=24) were freshly isolated from whole blood and stimulated for four hours with lipopolysaccharide (LPS). A methanol-based extraction protocol was developed and metabolomic profiling carried out using a Hydrophilic Interaction Liquid Chromatography (HILIC) LC-MS method. Data analysis pipelines used both targeted and untargeted approaches, and over 40 different data normalisation techniques to account for technical and biological variation were examined. Cytokine levels in supernatants were measured by ELISA.ResultsThis method provided broad coverage of the monocyte metabolome. The most efficient and consistent normalisation method was measurement of residual protein in the metabolite fraction, which was further validated and optimised using a commercial kit. Alterations to the monocyte metabolome in response to LPS can be detected as early as four hours post stimulation. Broad and profound changes in monocyte metabolism were seen, in line with increased cytokine production. Elevated levels of amino acids and Krebs cycle metabolites were noted and decreases in aspartate and β-alanine are also reported for the first time. In the untargeted analysis, 154 metabolite entities were significantly altered compared to unstimulated cells. Pathway analysis revealed the most prominent changes occurred to (phospho-) inositol metabolism, glycolysis, and the pentose phosphate pathway.DiscussionThese data report the emergent changes to monocyte metabolism in response to LPS, in line with reports from later time points. A number of these metabolites are reported to alter inflammatory gene expression, which may facilitate the increases in cytokine production. Further validation is needed to confirm the link between metabolic activation and upregulation of inflammatory responses.

免疫代谢学探讨免疫细胞功能与代谢之间的联系。免疫细胞代谢的失调现已成为先天免疫细胞激活的一个确立特征。液相色谱-质谱联用（LC-MS）技术在深入探究细胞代谢组学方面取得了显著进展。在本研究中，我们对文献中描述的多种样本制备技术和数据标准化方法进行了研究，并比较了它们在人类单核细胞代谢组学分析中的应用。方法：以脂多糖（LPS）刺激四小时后的原始单核细胞（n=24）作为研究模型。从全血中新鲜分离的单核细胞经脂多糖（LPS）刺激四小时。开发了一种基于甲醇的提取方案，并采用亲水性相互作用液相色谱-质谱（HILIC LC-MS）方法进行代谢组学分析。数据分析流程采用了靶向和非靶向方法，并考察了超过40种不同的数据标准化技术，以考虑技术性和生物学变异。通过酶联免疫吸附测定（ELISA）测量了上清液中的细胞因子水平。结果：该方法对单核细胞代谢组进行了广泛的覆盖。最有效和一致的标准化方法是测量代谢物组分中的残留蛋白质，该标准化方法进一步通过商业试剂盒进行了验证和优化。在刺激后四小时内即可检测到单核细胞代谢组对LPS的反应。观察到单核细胞代谢发生了广泛而深刻的改变，与细胞因子产生增加相一致。观察到氨基酸和三羧酸循环代谢物水平升高，同时首次报告了天冬氨酸和β-丙氨酸的降低。在非靶向分析中，与未刺激细胞相比，154种代谢物实体发生了显著改变。通路分析揭示了最显著的变化发生在（磷酸化）肌醇代谢、糖酵解和戊糖磷酸途径。讨论：这些数据报告了单核细胞代谢在LPS刺激下出现的早期变化，与后期时间点的报道相一致。其中许多代谢物被报道可以改变炎症基因的表达，这可能有助于细胞因子产生的增加。需要进一步验证以确认代谢激活与炎症反应上调之间的联系。