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 (1-48 h) 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 3 h after challenge with 1 μg/ml LPS, whereas TLR1 and TLR2 mRNA were increased (p<0.01) at 6 h after challenge with 10 μg/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 9 h and 1 h 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 1 h after LPS stimulation and remained constant till 48 h. IL12b was increased (p<0.001) 12 h after challenge with 10 μg/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.

Fig. 1.

Overall TLR1–10 mRNA expression patterns in LPS stimulated PBMCs. Data are expressed as the mean of all seven time points for a TLR within the same concentration of LPS.

Fig. 2.

TLR2, TLR4 and TLR6 mRNA expression in LPS stimulated PBMCs over time. Bar without common superscripts (lower case letter for control; capital-italic letter for 1 μg/ml LPS; and capital [non-italic] letter for 10 μg/ml LPS) differ (p < 0.05). Data are expressed as mean ± SD of duplicate samples; error bars for some conditions were so small they are obscured be the symbols. Relative mRNA expression of TLRs: (a) TLR2; (b) TLR4 and (c) TLR6. When LPS-stimulated mRNA expression values within a time point are compared with control: *p < 0.05, **p < 0.01, ***p < 0.001. Proc GLM data for each gene are shown on top-left corner.

Fig. 3.

TLR9, CD14 and MD2 mRNA expression in LPS stimulated PBMCs over time. Bar without common superscripts (lower case letter for control; capital-italic letter for 1 μg/ml LPS; and capital [non-italic] letter for 10 μg/ml LPS) differ (p < 0.05). Data are expressed as mean ± SD of duplicate samples; error bars for some conditions were so small they are obscured by the symbols. Relative mRNA expression of TLRs and associated molecules: (a) TLR9; (b) CD14 and (c) MD2. When LPS-stimulated mRNA expression values within a time point are compared with control: *p < 0.05, **p < 0.01, ***p < 0.001. Proc GLM data for each gene are shown on top-left corner.

Fig. 4.

Cytokines protein production by LPS stimulated PBMCs over time. Bar without common superscripts (lower case letter for control; capital-italic letter for 1 μg/ml LPS; and capital [non-italic] letter for 10 μg/ml LPS) differ (p < 0.05). Data are expressed as mean ± SD of duplicate samples; error bars for some conditions were so small they are obscured by the symbols. Cytokines protein (pg/ml) measured in the PBMCs culture supernatant: (a) IL1β; (b) IL8; (c) IL12 and (d) TNFα. When LPS-stimulated cytokines protein production values within a time point are compared with control: *p < 0.05, **p < 0.01, ***p < 0.001. Proc GLM data for each gene are shown on top-left corner.