332 Effects of Ruminal Lipopolysaccharides on Primary Bovine Ruminal Epithelial Cells

Abstract

Abstract The objective of this study was to investigate the immunopotential of mixed 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. In Exp. 1 the objective was to determine the immunopotential of ruminal LPS, in Exp. 2 the objective was to assess tolerance to chronic LPS exposure, and the objective of Exp. 3 was to evaluate antagonistic interactions between ruminal and E.coli LPS. In exp. 1 & 2, RECs were exposed to nonpyrogenic water (CON), 20 μg/mL of E. coli LPS (E. COLI), 10 μg/mL of mixed ruminal-LPS (RUM10), 20 μg/mL of mixed ruminal-LPS (RUM20) and 40 μg/mL of mixed 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 mixed ruminal-LPS:1 μg/mL E. coli LPS (1:1), 10 μg/mL mixed ruminal-LPS :1 μg/mL E. coli LPS (10:1) and 50 μg/mL mixed 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 a blocking factor. Each treatment had 2 technical replicates, and treatment responses for all data were analyzed with the MIXED procedure of SAS. For all exp., total RNA was extracted from RECs and real-time quantitative PCR was performed to determine the relative expression of genes for toll-like receptors (TLR2 & TLR4), proinflammatory cytokines (TNFα, IL1β, and IL6), chemokines (CXCL2 & CXCL8), growth factor-like cytokines (CSF2 & TGFβ), and a lipid mediator (PTGS2). In exp. 1, the targeted genes were upregulated in response to E. COLI (P<0.01), while all ruminal LPS treatments resulted in a lower transcript abundance (P<0.01). Regarding RPT and RCV condition, in Exp. 2, the expression of targeted genes was downregulated or not affected in response to ruminal LPS treatments. Lastly, in Exp. 3 all targeted genes resulted in decreased transcript abundance on all ruminal LPS ratios (P<0.01). Overall, our results indicate that ruminal LPS have a limited capacity to activate the TLR4/NF-κB pathway and to induce the expression of inflammatory genes.