Expression dynamics of Toll-like receptors mRNA and cytokines in porcine peripheral blood mononuclear cells stimulated by bacterial lipopolysaccharide

Abstract

The Toll-like receptor (TLR)4 is critical for the recognition of Gram-negative bacterial lipopolysaccharide (LPS) but in porcine peripheral blood mononuclear cells (PBMCs) it may cooperate with other TLRs and lead to the production of inflammatory cytokines. Therefore, we analyzed TLR1–10 mRNA expression in porcine PBMCs stimulated with LPS over time (1–48h) by using quantitative real-time PCR and cytokine proteins level by ELISA in culture supernatant. TLR1–10 mRNA was detectable in porcine PBMCs. When compared with the control (non-stimulated), TLR1 mRNA were increased (p<0.05) at 3h after challenge with 1μg/ml LPS, whereas TLR1 and TLR2 mRNA were increased (p<0.01) at 6h after challenge with 10μg/ml LPS. TLR4 increased (p<0.001) at 3h after challenge with LPS and remained constant. TLR5 and TLR6 mRNA increased (p<0.05) at 9h and 1h after of LPS stimulation, respectively. The mRNA of CD14 and MD2 were increased (p<0.001) at 1h after LPS stimulation. Additionally, at most of the time analyzed, the mRNA expression increased with the dose of LPS. The LPS concentration had influence (p<0.05) on all the TLRs expression except TLR10; whereas time had effect (p<0.05) on all TLRs expression except TLR2, 3, 6 and 10. When compared to the control, the cytokines IL1b, IL8 and TNFα proteins were increased (p<0.001) immediately at 1h after LPS stimulation and remained constant till 48h. IL12b was increased (p<0.001) 12h after challenge with 10μg/ml of LPS. Although IL8 level was the highest, the higher (p<0.05) expression of all these inflammatory cytokines indicate that upon interacting with TLRs, LPS exerted inflammatory response in PBMCs through the production of Th1 type cytokines. The production of cytokines was influenced (p<0.001) by both the dose of LPS and the stimulation time. Hence, the porcine PBMCs are likely able to express all members of TLRs.