Effects of lipopolysaccharide exposure in primary bovine ruminal epithelial cells

摘要

The objective of this study was to investigate whethercultured ruminal epithelial cells (REC) respondedto lipopolysaccharide (LPS) stimulation and determinewhether LPS induced a proinflammatory response. Primarybovine REC were isolated and grown in culture for2 studies. In study 1, REC were isolated from Holsteinbull calves (n = 8) and grown in culture for 10 to 12 d.Cells were then exposed to 0, 10,000, 50,000, or 200,000endotoxin (E)U/mL of LPS (Escherichia coli O55:B5)for either 6 or 24 h. The effect of LPS exposure on cellviability was analyzed by flow cytometry using a propidiumiodide stain. In study 2, cells were isolated fromHolstein 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) mediumalone 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 for24 h before restimulating with LPS for an additional12 h (RPT), and (6) 12 h of LPS exposure followedby LPS removal for 36 (RVY). For both experiments,total RNA was extracted from REC and real-timequantitative PCR was performed to determine relativeexpression of genes for toll-like receptors (TLR2 andTLR4), proinflammatory cytokines (TNF and IL1B),chemokines (CXCL2 and CXCL8), a lipid mediator(PTGS2), and growth factor-like cytokines (CSF2 andIL7). In study 1, LPS exposure did not negatively affectcell viability. Treatment of cells with LPS resulted inincreased transcript abundance for all genes analyzed.The TLR2, IL7, and TLR4 had a greater magnitude ofchange at 6 h compared with 24 h. Quadratic expressionpatterns were detected for TNF, IL1B, CXCL2,CXCL8, and CSF2. These results suggested that RECincrease expression of proinflammatory genes followingexposure to LPS. In study 2, all genes analyzed wereupregulated in a quadratic manner following exposureto LPS for different time intervals. The TLR4, TNF,CXCL2, CXCL8, CSF2, and IL7 gene expression wassignificantly greater after a single 12 h of LPS exposurethan after RPT exposure, suggesting repeated exposureof REC to LPS may induce a tolerogenic effect. WhenLPS was removed from the medium (RVY), transcriptabundance for all genes analyzed decreased and expressionof TLR2, TLR4, and IL7 returned to baselinelevels, suggesting REC recovered following exposure toLPS. Overall, the data suggest cultured REC respondto LPS stimulation by increasing transcription of proinflammatorygenes and this transcriptional responsewas influenced by the dose, duration, and frequency ofLPS exposure.