| Abstrakt: |
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.coliLPS. In exp. 1 & 2, RECs were exposed to nonpyrogenic water (CON), 20 μg/mL of E. coliLPS (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. coliLPS (0:1), 1 μg/mL mixed ruminal-LPS:1 μg/mL E. coliLPS (1:1), 10 μg/mL mixed ruminal-LPS :1 μg/mL E. coliLPS (10:1) and 50 μg/mL mixed ruminal-LPS :1 μg/mL E. coliLPS (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. |
