摘要
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.
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 (IL1B), 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.
基金
provided by the Future Interdisciplinary Research Explorations grant program of the Office of Research,College of ACES,University of Illinois at Urbana-Champaign,through the USDA National Institute of Food and Agriculture Hatch project ILLU-538-395(Accession Number 0232734)and ILLU-538-914
