Effects of lipopolysaccharide exposure in primary bovine ruminal epithelial cells

Abstract

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.