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

Omar Bulgari; Xianwen Dong; Alfred L.Roca; Anna M.Caroli; Juan J.Loor

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 (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.

机译：背景：使用革兰氏阴性脂多糖（LPS）和革兰氏阳性脂蛋白酸（LTA）细菌细胞壁成分体外刺激初级乳腺上皮细胞（MEC）诱导的先天免疫应答在牛物种中具有良好的特征。当前的研究是为了表征原代山羊MEC（pgMEC）中Toll样受体诱导的炎症反应的下游调节。我们进行了定量实时RT-PCR（qPCR），以测量参与转录调节的9个基因的mRNA水平或抗菌活性：Toll样受体2（TLR2），Toll样受体4（TLR4），前列腺素-过氧化物过氧化物合酶2（PTGS2），干扰素诱导的四肽重复序列3（IFIT3），干扰素调节因子3（IRF3），髓样分化初级反应88（MYD88），B细胞1中κ轻型多肽基因增强子的核因子（NFKB1），Toll相互作用蛋白（TOLLIP）和乳铁蛋白（LTF）。第7类细胞因子参与了Toll样受体信号通路：CC基序趋化因子配体2（CCL2），CC基序趋化因子配体5（CCL5），CXC基序趋化因子配体6（CXCL6），白介素8（CXCL8），白介素1 beta（IL1B）），白介素6（IL6）和肿瘤坏死因子α（TNF）。结果：LPS刺激pgMEC 3 h导致CCL2，CXCL6，IL6，CXCL8，PTGS2，IFIT3，MYD88，NFKB1， LPS后6 h，除IL6和PTGS2外，相同基因的表达均高于对照组（P ＜0.05）。与LTA孵育3小时后，与对照相比更大（P ＜0.05）。与对照组相比，用LTA刺激6 h导致PTGS2，IFIT3，NFKB1和TOLLIP的表达更高（P ＜0.05），而CXCL6， CXCL8和TLR4较低（P ＜0.05）。与对照相比，两种毒素孵育3 h CCL2，CCL5，CXCL6，CXCL8，IL6，PTGS2，IFIT3，IRF3，MYD88和NFKB1被检测到d。两种毒素孵育6 h后，CCL2，CXCL6，IFIT3，MYD88，NFKB1和TLR4的表达均高于对照组（P ＜0.05）。炎症反应比LPS弱。这可能与以下事实有关：革兰氏阳性细菌比革兰氏阴性感染更容易引起慢性乳腺炎。

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

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