While feed components capable of modulating the immune system are highly sought after and marketed, often little evidence is available to support functional immune response claims. Thus, a high-throughput in vitro cell screening system was developed to test these compounds for innate immune signaling effects, using Saccharomyces cerevisiae and its cell wall components in addition to lauric acid and its esters as models in two separate experiments. This screening system utilized RAW 264.7 murine macrophages to assess live S. cerevisiae cells and S. cerevisiae-derived cell wall components β-glucan, mannan, and zymosan (a crude cell wall preparation containing both β-glucan and mannan). D-mannose was also evaluated as the monomer of mannan. We also examined the effect of a saturated fatty acid (C12:0, lauric acid) and its esters (methyl laurate and glycerol monolaurate) on innate immune cell activation and cellular metabolism. RAW cells were transfected with a vector that drives expression of alkaline phosphatase upon promoter activation of nuclear factor κ-light-chain-enhancer of activated B cells (NFκB), a major inflammatory/immune transcription factor. RAW cells were incubated with 0.01, 0.1 or 1 mg/mL of yeast compounds alone or RAW cells were challenged with LPS and then incubated with yeast compounds. In a separate experiment, RAW cells were incubated with 0, 0.5, 2.5, 12.5, 62.5, and 312.5 μmol/L of lauric acid, methyl laurate, or glycerol monolaurate alone, or RAW cells were challenged with LPS and then incubated with fatty acid treatments. Treatment with zymosan or β-glucan alone induced NFκB activation in a dose-dependent manner, whereas treatment with D-mannose, mannan, or live S. cerevisiae cells did not. Post-treatment with mannan after an LPS challenge decreased NFκB activation, suggesting that this treatment may ameliorate LPS-induced inflammation. Slight increases in NFκB activation were found when fatty acid treatments were applied in the absence of LPS, yet substantial reductions in NFκB activation were seen when treatments were applied following an LPS challenge. Overall, this cell screening system using RAW macrophages was effective, high-throughput, and sensitive to feed components combined with LPS challenges, indicating modulation of innate immune signaling in vitro.

中文翻译：

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饲料成分免疫调节作用的体外巨噬细胞筛选系统的开发。

虽然能够调节免疫系统的饲料成分受到高度追捧和销售，但通常很少有证据支持功能性免疫反应声称。因此，开发了一种高通量体外细胞筛选系统来测试这些化合物的先天免疫信号效应，在两个独立的实验中使用酿酒酵母及其细胞壁成分以及月桂酸及其酯作为模型。该筛选系统利用 RAW 264.7 鼠巨噬细胞来评估活的酿酒酵母细胞和酿酒酵母衍生的细胞壁成分 β-葡聚糖、甘露聚糖和酵母聚糖（一种含有 β-葡聚糖和甘露聚糖的粗制细胞壁制剂）。D-甘露糖也被评价为甘露聚糖的单体。我们还检查了饱和脂肪酸 (C12:0, 月桂酸）及其酯（月桂酸甲酯和单月桂酸甘油酯）对先天免疫细胞活化和细胞代谢的影响。用一种载体转染 RAW 细胞，该载体在激活 B 细胞的核因子 κ-轻链增强子 (NFκB)（一种主要的炎症/免疫转录因子）的启动子激活后驱动碱性磷酸酶的表达。RAW 细胞单独与 0.01、0.1 或 1 mg/mL 酵母化合物一起孵育，或者 RAW 细胞用 LPS 攻击，然后与酵母化合物一起孵育。在单独的实验中，RAW 细胞与 0、0.5、2.5、12.5、62.5 和 312.5 μmol/L 的月桂酸、月桂酸甲酯或单月桂酸甘油酯单独孵育，或者 RAW 细胞先用 LPS 攻击，然后与脂肪酸孵育治疗。酵母聚糖或β-葡聚糖单独治疗以剂量依赖性方式诱导NFκB活化，而用 D-甘露糖、甘露聚糖或活的酿酒酵母细胞处理则没有。在 LPS 攻击后用甘露聚糖进行后处理降低了 NFκB 的活化，表明这种治疗可能会改善 LPS 诱导的炎症。当在没有 LPS 的情况下应用脂肪酸处理时，发现 NFκB 活化略有增加，但在 LPS 攻击后应用处理时，NFκB 活化显着降低。总体而言，这种使用 RAW 巨噬细胞的细胞筛选系统是有效的、高通量的，并且对结合 LPS 挑战的饲料成分敏感，表明体外先天免疫信号传导的调节。表明这种治疗可以改善 LPS 诱导的炎症。当在没有 LPS 的情况下应用脂肪酸处理时，发现 NFκB 活化略有增加，但在 LPS 攻击后应用处理时，NFκB 活化显着降低。总体而言，这种使用 RAW 巨噬细胞的细胞筛选系统是有效的、高通量的，并且对结合 LPS 挑战的饲料成分敏感，表明体外先天免疫信号传导的调节。表明这种治疗可以改善 LPS 诱导的炎症。当在没有 LPS 的情况下应用脂肪酸处理时，发现 NFκB 活化略有增加，但在 LPS 攻击后应用处理时，NFκB 活化显着降低。总体而言，这种使用 RAW 巨噬细胞的细胞筛选系统是有效的、高通量的，并且对结合 LPS 挑战的饲料成分敏感，表明体外先天免疫信号传导的调节。