Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5A8.
Institute of Veterinary Physiology, Freie Universität Berlin, D-14163, Berlin, Germany.
J Dairy Sci. 2020 Oct;103(10):9587-9603. doi: 10.3168/jds.2020-18652. Epub 2020 Jul 31.
The objective of this study was to investigate whether cultured ruminal epithelial cells (REC) responded to lipopolysaccharide (LPS) stimulation and determine whether LPS induced a proinflammatory response. Primary bovine REC were isolated and grown in culture for 2 studies. In study 1, REC were isolated from Holstein bull calves (n = 8) and grown in culture for 10 to 12 d. Cells were then exposed to 0, 10,000, 50,000, or 200,000 endotoxin (E)U/mL of LPS (Escherichia coli O55:B5) for either 6 or 24 h. The effect of LPS exposure on cell viability was analyzed by flow cytometry using a propidium iodide stain. In study 2, cells were isolated from Holstein bull calves (n = 5) and yearling beef heifers (n = 4). Cells were exposed to either 1,000 or 50,000 EU/mL of LPS using the following conditions: (1) medium alone time-matched controls, (2) 12-h LPS exposure, (3) 24 h of LPS exposure, (4) 36 h of LPS exposure, (5) 12 h of LPS exposure followed by LPS removal for 24 h before restimulating with LPS for an additional 12 h (RPT), and (6) 12 h of LPS exposure followed by LPS removal for 36 (RVY). For both experiments, total RNA was extracted from REC and real-time quantitative PCR was performed to determine relative expression of genes for toll-like receptors (TLR2 and TLR4), proinflammatory cytokines (TNF and IL1B), chemokines (CXCL2 and CXCL8), a lipid mediator (PTGS2), and growth factor-like cytokines (CSF2 and IL7). In study 1, LPS exposure did not negatively affect cell viability. Treatment of cells with LPS resulted in increased transcript abundance for all genes analyzed. The TLR2, IL7, and TLR4 had a greater magnitude of change at 6 h compared with 24 h. Quadratic expression patterns were detected for TNF, IL1B, CXCL2, CXCL8, and CSF2. These results suggested that REC increase expression of proinflammatory genes following exposure to LPS. In study 2, all genes analyzed were upregulated in a quadratic manner following exposure to LPS for different time intervals. The TLR4, TNF, CXCL2, CXCL8, CSF2, and IL7 gene expression was significantly greater after a single 12 h of LPS exposure than after RPT exposure, suggesting repeated exposure of REC to LPS may induce a tolerogenic effect. When LPS was removed from the medium (RVY), transcript abundance for all genes analyzed decreased and expression of TLR2, TLR4, and IL7 returned to baseline levels, suggesting REC recovered following exposure to LPS. Overall, the data suggest cultured REC respond to LPS stimulation by increasing transcription of proinflammatory genes and this transcriptional response was influenced by the dose, duration, and frequency of LPS exposure.
本研究旨在探讨培养的瘤胃上皮细胞（REC）是否对脂多糖（LPS）刺激有反应，并确定 LPS 是否诱导了促炎反应。从荷斯坦公牛犊（n = 8）中分离和培养原代牛 REC 进行了 2 项研究。在研究 1 中，将 REC 培养 10 至 12 天，然后用 0、10,000、50,000 或 200,000 内毒素（E）U/mL LPS（大肠杆菌 O55:B5）分别孵育 6 或 24 小时。用碘化丙啶染色通过流式细胞术分析 LPS 暴露对细胞活力的影响。在研究 2 中，从荷斯坦公牛犊（n = 5）和青年肉牛（n = 4）中分离细胞。将细胞用 1,000 或 50,000 EU/mL LPS 孵育，条件如下：（1）仅培养基对照，时间匹配；（2）12 小时 LPS 暴露；（3）24 小时 LPS 暴露；（4）36 小时 LPS 暴露；（5）12 小时 LPS 暴露，然后去除 LPS 24 小时，然后再用 LPS 刺激 12 小时（RPT）；（6）12 小时 LPS 暴露，然后去除 LPS 36 小时（RVY）。对于这两个实验，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 和 IL1B）、趋化因子（CXCL2 和 CXCL8）、脂质介质（PTGS2）和生长因子样细胞因子（CSF2 和 IL7）的基因相对表达。在研究 1 中，LPS 暴露不会对细胞活力产生负面影响。用 LPS 处理细胞会导致所有分析基因的转录物丰度增加。TLR2、IL7 和 TLR4 在 6 小时的变化幅度大于 24 小时。TNF、IL1B、CXCL2、CXCL8 和 CSF2 检测到二次表达模式。这些结果表明，REC 在 LPS 暴露后增加了促炎基因的表达。在研究 2 中，在不同的 LPS 孵育时间后，所有分析的基因都以二次方式上调。与 RPT 暴露相比，单次 12 小时 LPS 暴露后 TLR4、TNF、CXCL2、CXCL8、CSF2 和 IL7 的基因表达显著更高，表明 REC 反复暴露于 LPS 可能会诱导耐受作用。当 LPS 从培养基中去除（RVY）时，所有分析基因的转录物丰度降低，TLR2、TLR4 和 IL7 的表达恢复到基线水平，表明 REC 在暴露于 LPS 后得到了恢复。总体而言，数据表明培养的 REC 通过增加促炎基因的转录对 LPS 刺激做出反应，这种转录反应受 LPS 剂量、持续时间和频率的影响。
