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Evaluation of Porcine IPEC-J2 Cell Line Immune Response to Escherichia coli (0111:B4) Lipopolysaccharide
The objective of this study was to evaluate expression of genes associated with the proinflammatory immune response in IPEC-J2 cell line after exposure to pathogenic E. coli O111:B4-derived lipopolysaccharide (LPS). Upon confluence, IPEC-J2 cells were cultured in a penicillin-streptomycin-free medium for a week prior to LPS challenge. Afterward, cells were challenged with LPS based on a 4×5 factorial arrangement consisting of four levels of LPS (0, 0.1, 1, and 10 μg/mL) and 5 exposure durations (0, 1, 2, 4, and 6 h). At the end of the challenge, cells were lysed with TRIzol reagent and total RNA was extracted. Gene expression levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, -6, -8, -10, toll-like receptor (TLR) -1, -2, -3, -4, -6, and -8 were evaluated using a two-step reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR). No interaction between LPS concentration and culture time among genes was observed (P > 0.10). Results of contrast analysis showed that expression of TNF-α (Relative quantity = 0.82, 1.66, 2.12 and 2.70 at 0, 0.1, 1, and 10 μg/mL LPS; P = 0.10) and TLR-8 (0.61, 0.65, 0.74, 0.94; P = 0.05) was upregulated linearly with increasing LPS concentration up to 10 μg/mL. There was a tendency for quadratic increases of IL-8 (3.15, 4.18, 5.82, 5.39; P = 0.09) with greatest expression at 1 μg/mL LPS. Expression of GM-CSF (0.91 7.78, 13.12, 4.45 and 3.52 at 0, 1, 2, 4, and 6 h, respectively), IL-8 (0.60, 3.71, 7.03, 7.07, 4.77), and TLR4 (0.52, 0.43, 0.61, 0.95, 0.58) was significantly stimulated at longer culture times (Quadratic; P < 0.05), peaking at 2, 4, and 4 h after challenge, respectively. In addition, mRNA levels of TLR-2 (0.65, 1.00, 1.03, 1.13, 0.79) and -6 (0.71, 0.74, 1.11, 1.08, 0.64) tended to increase at extended culture times (Quadratic; P ≤ 0.10) with the greatest levels at 4 and 2 h, respectively. TLR-3 (0.60, 0.40, 0.34, 0.46, 0.41), however, tended to decrease (Quadratic; P ≤ 0.10) with extended culture time and reached the lowest level at 4 h. Expression of IL-6 decreased linearly (0.60, 0.55, 0.32, 0.26, 0.32; P < 0.05) as culture time increased. Results of the proinflammatory response when IPEC-J2 cell line under LPS challenge in current study indicate that the IPEC-J2 cell line may be used as an in vitromodel to evaluate the impact of treatment on immune response.
Keywords: IPEC-J2, immunity, gene expression