Bulgari Omar, Dong Xianwen, Roca Alfred L, Caroli Anna M, Loor Juan J
Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.
Department of Molecular and Translational Medicine, University of Brescia, Brescia, 25123 Italy.
J Anim Sci Biotechnol. 2017 Apr 1;8:29. doi: 10.1186/s40104-017-0162-8. eCollection 2017.
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 (), Toll-like receptor 4 (), prostaglandin-endoperoxide synthase 2 (), interferon induced protein with tetratricopeptide repeats 3 (), interferon regulatory factor 3 (), myeloid differentiation primary response 88 (), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (), Toll interacting protein (), and lactoferrin (). Furthermore, we analyzed 7 cytokines involved in Toll-like receptor signaling pathways: C-C motif chemokine ligand 2 (), C-C motif chemokine ligand 5 (), C-X-C motif chemokine ligand 6 (), interleukin 8 (), interleukin 1 beta (), interleukin 6 (), and tumor necrosis factor alpha ().
Stimulation of pgMEC with LPS for 3 h led to an increase in expression of , , , , , , , , and ( < 0.05). Except for , and , the same genes had greater expression than controls at 6 h post-LPS ( < 0.05). Expression of , , , , , and was greater than controls after 3 h of incubation with LTA ( < 0.05). Compared to controls, stimulation with LTA for 6 h led to greater expression of , , , and ( < 0.05) whereas the expression of , , and was lower ( < 0.05). At 3 h incubation with both toxins compared to controls a greater expression ( < 0.05) of , , , , , , , , , and was detected. After 6 h of incubation with both toxins, the expression of , , , , , and was higher than the controls ( < 0.05).
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.
利用革兰氏阴性脂多糖（LPS）和革兰氏阳性脂磷壁酸（LTA）细菌细胞壁成分对原代乳腺上皮细胞（MEC）进行体外刺激所诱导的先天性免疫反应，在牛种中已有充分的特征描述。本研究的目的是表征原代山羊MEC（pgMEC）中Toll样受体诱导的炎症反应的下游调控。我们进行了定量实时RT-PCR（qPCR），以测量参与转录调控或抗菌活性的9个基因的mRNA水平：Toll样受体2（）、Toll样受体4（）、前列腺素内过氧化物合酶2（）、含四肽重复序列的干扰素诱导蛋白3（）、干扰素调节因子3（）、髓样分化初级反应88（）、B细胞中κ轻链多肽基因增强子的核因子1（）、Toll相互作用蛋白（）和乳铁蛋白（）。此外，我们分析了参与Toll样受体信号通路的7种细胞因子：C-C基序趋化因子配体2（）、C-C基序趋化因子配体5（）、C-X-C基序趋化因子配体6（）、白细胞介素8（）、白细胞介素1β（）、白细胞介素6（）和肿瘤坏死因子α（）。
用LPS刺激pgMEC 3小时导致、、、、、、、和的表达增加（<0.05）。除了、和外，相同基因在LPS处理后6小时的表达高于对照组（<0.05）。用LTA孵育3小时后，、、、、和的表达高于对照组（<0.05）。与对照组相比，用LTA刺激6小时导致、、和的表达增加（<0.05），而、和的表达降低（<0.05）。与对照组相比，在用两种毒素孵育3小时时，检测到、、、、、、、、和的表达更高（<0.05）。在用两种毒素孵育6小时后，、、、、和的表达高于对照组（<0.05）。
数据表明，在山羊MEC中，LTA诱导的炎症反应比LPS弱。这可能与革兰氏阳性菌比革兰氏阴性菌感染更常引起慢性乳腺炎的观察结果有关。
