Effects of ruminal LPS exposure on primary bovine ruminal epithelial cells.

The objective of this study was to investigate the immunopotential of ruminal lipopolysaccharides (LPS) on cultured primary bovine ruminal epithelial cells (RECs). Primary bovine RECs 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 E.coli LPS. In experiments 1 and 2, RECs were exposed to nonpyrogenic water (CON), 20 μg/mL of E. coli LPS (E.COLI), 10 μg/mL of ruminal LPS (RUM10), 20 μg/mL of ruminal LPS (RUM20), and 40 μg/mL of ruminal LPS (RUM40), either continuously or intermittently. For the continuous exposure, RECs underwent a 6 h exposure, while 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 exp 3, RECs were exposed to nonpyrogenic water (0-0), 1 μg/mL E. coli LPS (0-1), 1 μg/mL ruminal LPS:1 μg/mL E. coli LPS (1-1), 10 μg/mL ruminal LPS:1 μg/mL E. coli LPS (10-1) and 50 μg/mL ruminal LPS:1 μg/mL E. coli LPS (50-1). 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 RECs and real-time qPCR 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 E.COLI, while 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 E.COLI 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.

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© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).