Department of Animal Sciences, University of Florida, Gainesville, FL 32608.
Department of Animal Sciences, University of Florida, Gainesville, FL 32608.
J Dairy Sci. 2024 Feb;107(2):1244-1262. doi: 10.3168/jds.2023-23736. Epub 2023 Sep 29.
The objective of this study was to investigate the immunopotential of ruminal lipopolysaccharides (LPS) on cultured primary bovine rumen epithelial cells (REC). Primary bovine REC were isolated from 6 yearling steers and grown in culture for 3 experiments. Experiment 1 aimed to determine the immunopotential of ruminal LPS, experiment 2 aimed to assess tolerance to chronic LPS exposure, and experiment 3 aimed to evaluate antagonistic interactions between ruminal and Escherichia coli LPS. In experiments 1 and 2, REC were exposed to nonpyrogenic water, 20 μg/mL E. coli LPS (EC20), 10 μg/mL ruminal LPS, 20 μg/mL ruminal LPS, and 40 μg/mL ruminal LPS, either continuously or intermittently. For the continuous exposure, REC underwent a 6 h exposure, whereas for the intermittent exposure, the procedure was: (1) a 12 h continuous exposure to treatments followed by LPS removal for 24 h and then another 12 h of exposure (RPT), and (2) a 12 h continuous exposure to treatments followed by LPS removal and a recovery period of 36 h (RCV). In experiment 3, REC were exposed to nonpyrogenic water, 1 μg/mL E. coli LPS, 1 μg/mL ruminal LPS to 1 μg/mL E. coli LPS, 10 μg/mL ruminal LPS to 1 μg/mL E. coli LPS, and 50 μg/mL ruminal LPS to 1 μg/mL E. coli LPS. Each experiment was done as a complete randomized block design with 6 REC donors. The REC-donor was used as blocking factor. Each treatment had 2 technical replicates, and treatment responses for all data were analyzed with the MIXED procedure of SAS. For all experiments, total RNA was extracted from REC and real-time quantitative PCR was performed to determine the relative expression of genes for toll-like receptors (TLR2 and TLR4), proinflammatory cytokines (TNF, IL1B, and IL6), chemokines (CXCL2 and CXCL8), growth factor-like cytokines (CSF2 and TGFB1), and a lipid mediator (PTGS2). In experiment 1, the targeted genes were upregulated by EC20, whereas all ruminal LPS treatments resulted in a lower transcript abundance. Regarding RPT, and RCV condition, in experiment 2, the expression of targeted genes was not affected or was at a lower abundance to EC20 when compared with ruminal LPS treatments. Lastly, in experiment 3, all targeted genes resulted in lower or similar transcript abundance on all ruminal LPS ratios. Overall, our results indicate that ruminal LPS have a limited capacity to activate the TLR4/NF-kB pathway and to induce the expression of inflammatory genes.
本研究旨在探讨瘤胃脂多糖（LPS）对培养的原代牛瘤胃上皮细胞（REC）的免疫潜力。原代牛 REC 从 6 头育肥牛中分离出来，并在培养物中生长了 3 次实验。实验 1 旨在确定瘤胃 LPS 的免疫潜力，实验 2 旨在评估对慢性 LPS 暴露的耐受性，实验 3 旨在评估瘤胃和大肠杆菌 LPS 之间的拮抗相互作用。在实验 1 和 2 中，REC 暴露于非热原性水、20μg/mL 大肠杆菌 LPS（EC20）、10μg/mL 瘤胃 LPS、20μg/mL 瘤胃 LPS 和 40μg/mL 瘤胃 LPS 中，无论是连续暴露还是间歇暴露。对于连续暴露，REC 经历了 6 小时暴露，而对于间歇暴露，过程为：（1）连续 12 小时暴露于处理物，然后去除 LPS 24 小时，然后再暴露 12 小时（RPT），和（2）连续 12 小时暴露于处理物，然后去除 LPS 并进行 36 小时的恢复（RCV）。在实验 3 中，REC 暴露于非热原性水、1μg/mL 大肠杆菌 LPS、1μg/mL 瘤胃 LPS 至 1μg/mL 大肠杆菌 LPS、10μg/mL 瘤胃 LPS 至 1μg/mL 大肠杆菌 LPS 和 50μg/mL 瘤胃 LPS 至 1μg/mL 大肠杆菌 LPS。每个实验都采用完全随机区组设计，有 6 个 REC 供体。REC-供体用作分组因素。每个处理有 2 个技术重复，所有数据的处理响应均使用 SAS 的 MIXED 程序进行分析。对于所有实验，从 REC 中提取总 RNA，并进行实时定量 PCR，以确定 Toll 样受体（TLR2 和 TLR4）、促炎细胞因子（TNF、IL1B 和 IL6）、趋化因子（CXCL2 和 CXCL8）、生长因子样细胞因子（CSF2 和 TGFB1）和脂质介质（PTGS2）的相对表达。在实验 1 中，EC20 上调了靶向基因，而所有瘤胃 LPS 处理均导致转录物丰度降低。关于 RPT 和 RCV 条件，在实验 2 中，与瘤胃 LPS 处理相比，靶向基因的表达不受影响或丰度较低。最后，在实验 3 中，所有靶向基因在所有瘤胃 LPS 比值下的转录物丰度均较低或相似。总体而言，我们的结果表明，瘤胃 LPS 激活 TLR4/NF-κB 途径和诱导炎症基因表达的能力有限。
