Innate immune responses induced by lipopolysaccharide and lipoteichoic acid in primary goat mammary epithelial cells

Background: Innate immune responses induced by in vitro stimulation of primary mammary epithelial cells(MEC) using Gram-negative lipopolysaccharide(LPS) and Gram-positive lipoteichoic acid(LTA) bacterial cell wall components are well-characterized in bovine species. The objective of the current study was to characterize the downstream regulation of the inflammatory response induced by Toll-like receptors in primary goat MEC(pgMEC). We performed quantitative real-time RT-PCR(qPCR) to measure mRNA levels of 9 genes involved in transcriptional regulation or antibacterial activity: Toll-like receptor 2(TLR2), Toll-like receptor 4(TLR4), prostaglandin-endoperoxide synthase 2(PTGS2), interferon induced protein with tetratricopeptide repeats 3(IFIT3), interferon regulatory factor 3(IRF3), myeloid differentiation primary response 88(MYD88), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1(NFKB1), Toll interacting protein(TOLLIP), and lactoferrin(LTF). Furthermore,we analyzed 7 cytokines involved in Toll-like receptor signaling pathways: C-C motif chemokine ligand 2(CCL2), C-C motif chemokine ligand 5(CCL5), C-X-C motif chemokine ligand 6(CXCL6), interleukin 8(CXCL8), interleukin 1 beta(IL1 B), interleukin 6(IL6), and tumor necrosis factor alpha(TNF).Results: Stimulation of pgMEC with LPS for 3 h led to an increase in expression of CCL2, CXCL6, IL6, CXCL8,PTGS2, IFIT3, MYD88, NFKB1, and TLR4(P < 0.05). Except for IL6, and PTGS2, the same genes had greater expression than controls at 6 h post-LPS(P < 0.05). Expression of CCL5, PTGS2, IFIT3, NFKB1, TLR4, and TOLLIP was greater than controls after 3 h of incubation with LTA(P < 0.05). Compared to controls, stimulation with LTA for 6 h led to greater expression of PTGS2, IFIT3, NFKB1, and TOLLIP(P < 0.05) whereas the expression of CXCL6, CXCL8, and TLR4 was lower(P < 0.05). At 3 h incubation with both toxins compared to controls a greater expression(P < 0.05) of CCL2, CCL5, CXCL6, CXCL8, IL6, PTGS2, IFIT3, IRF3, MYD88, and NFKB1 was detected. After 6 h of incubation with both toxins, the expression of CCL2, CXCL6, IFIT3, MYD88, NFKB1, and TLR4 was higher than the controls(P < 0.05).Conclusions: Data indicate that in the goat MEC, LTA induces a weaker inflammatory response than LPS.This may be related to the observation that gram-positive bacteria cause chronic mastitis more often than gram-negative infections.