Development of a simple typing method for the ovine Toll-like receptor 4 gene

Toll-like receptor 4 (TLR4) recognizes lipopolysaccharide (LPS) from Gram-negative bacteria, as well as a number of other ligands. Genetic variation in the TLR gene has been associated with altered immune responses to pathogens and results in variation in disease susceptibility. The objective of this work was to develop a simple and rapid genotyping system for ovine TLR4 and of a sensitivity that would allow detection of allelic variation in this gene. While variation in exon 3 of the ovine TLR4 gene has been described previously, we describe here an improved genotyping method. This method could not only reveal the four alleles that have been reported previously, but also revealed a further three new alleles of this gene in a population of 1670 New Zealand sheep. This improved genotyping method will be useful in understanding innate immune responses in individual sheep and could also be a useful tool for large-scale immune studies in sheep production systems.     

内毒素的生物学特性及其对非洲猪瘟流行下猪只复养的影响

文章就内毒素的生物学特性及其对非洲猪瘟(ASF)流行下的复养猪只健康的影响进行了综述,指出:在当前ASF流行下,认识内毒素(ET)的生物学特性及其对复养猪只健康的影响,近期意义在于猪场的成功复养与成功“拔牙”,以维持猪只的健康生产;远期意义在于开发出有效的细菌ET净化剂,这是一个在人医和兽医学领域所面临的极富挑战性的前沿性研究课题。     

脂多糖对小鼠子宫内膜Toll样受体4介导的炎性信号通路的影响

革兰阴性菌感染可引起子宫内膜炎,机体的Toll样受体4(TLR4)可接受革兰阴性菌的细胞壁主要成分脂多糖(LPS)的刺激引发一系列炎症反应。为了研究LPS对TLR4介导的炎性信号通路的影响,本试验以小鼠为模型,分别用不同浓度的LPS对小鼠进行在体子宫灌注和处理体外培养的子宫内膜上皮细胞系。组织学观察显示,灌注不同浓度LPS后子宫内膜组织中炎性细胞增多。通过RT-PCR对各组中TLR4和核转录因子κB(NF-κB)、IL-6的mRNA表达水平进行检测,发现LPS刺激能够增强TLR4和NF-κB、IL-6 mRNA表达,影响TLR4介导的炎性信号通路。     

Staphylococcus aureus and Escherichia coli elicit different innate immune responses from bovine mammary epithelial cells

Escherichia coli and Staphylococcus aureus are the most important pathogenic bacteria causing bovine clinical mastitis and subclinical mastitis, respectively. However, little is known about the molecular mechanisms underlying the different host response patterns caused by these bacteria. The aim of this study was to characterize the different innate immune responses of bovine mammary epithelium cells (MECs) to heat-inactivated E. coli and S. aureus. Gene expression of Toll-like receptor 2 (TLR2) and TLR4 was compared. The activation of nuclear factor kappa B (NF-κB) and the kinetics and levels of cytokine production were analyzed. The results show that the mRNA for TLR2 and TLR4 was up-regulated when the bovine MECs were stimulated with heat-inactivated E. coli, while only TLR2 mRNA was up-regulated when the bovine MECs were stimulated with heat-inactivated S. aureus. The expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 increased more rapidly and higher when the bovine MECs were stimulated with heat-inactivated E. coli than when they were stimulated with heat-inactivated S. aureus. E. coli strongly activated NF-κB in the bovine MECs, while S. aureus failed to activate NF-κB. Heat-inactivated S. aureus could induce NF-κB activation when bovine MECs cultured in medium without fetal calf serum. These results were confirmed using TLR2- and TLR4/MD2-transfected HEK293 cells and suggested that differential TLR recognition and the lack of NF-κB activation account for the impaired immune response elicited by heat-inactivated S. aureus.     

山羊TLR2绿色荧光蛋白融合载体的构建

为了构建在哺乳动物细胞中表达的山羊TLR2绿色荧光蛋白融合载体,试验根据克隆到T载体上的山羊TLR2测序结果,设计1对不含终止子的引物,将山羊TLR2 PCR产物连接到pEGFP-N1载体上,用磷酸钙法转染293T细胞,并在荧光显微镜下观察。结果表明:重组质粒经酶切和测序鉴定正确,且在293T细胞中表达;融合蛋白发出绿色荧光,表明TLR2-EGFP主要分布在细胞膜上。说明试验成功地构建了pEGFP-TLR2-N1绿色荧光蛋白融合载体。     

TLR2基因多态性与奶牛体细胞评分的相关性研究

旨在分析Toll-样受体2在奶牛隐性乳房炎中的作用。本研究以同一牛场内的中国荷斯坦奶牛和新疆褐牛为研究对象,选取奶牛体细胞数小于20万和大于100万的个体各15头,对TLR2基因进行测序,然后对发现的多态位点进行PCR-RFLP检测,分析这些位点与体细胞评分(SCS)的相关性。结果发现了TLR2基因E+189、E+631和E+2260 3个SNP位点,其中E+631和E+2260位点为本试验首次发现。E+189、E+631和E+2260 3个SNP位点在2个品种内均处于Hardy-Weinberg平衡状态(P>0.05);3个位点在2个品种间的基因型分布差异都极显著(P<0.01)。分析每个SNP位点与SCS的相关性,显示TLR2E+189位点AA比BB和AB基因型个体的SCS高(P<0.05),AB与BB基因型个体的SCS差异不显著(P>0.05);说明BB基因型个体的乳房炎发病率低。而TLR2E+631的SCS和TLR2E+189位点表现相似,但各个基因型个体间的SCS差异不显著(P>0.05)。E+2260位点AA基因型个体比AB的SCS低(P>0.05)。新疆褐牛的SCS低于荷斯坦牛(P<0.01),...     

Emodin reduces hypoglycemia/hypoxia-induced injury of microglia by TLR4/NF-κB signaling pathway

AIM: To investigate the mechanism of emodin on the protection of glucose-deficient/anoxic microglia. METHODS: A microglia BV2 cell model induced by hypoglycemia/hypoxia (HH) was established. The glucose-deficient/anoxic cells treated with emodin were labeled as HH+emodin (20, 40 and 80 μmol/L) groups. The BV2 cells with TLR4 over-expression treated with emodin under hypoglycemia/hypoxia condition was labeled as HH+pcDNA-TLR4+ emodin (40 μmol/L) group. The cell viability was measured by MTT assay. Lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) levels were detected by ELISA. The apoptosis was analyzed by flow cytometry. The protein levels of Bax, Bcl-2, TLR4, p-IκB and IκB were determined by Western blot. RESULTS: Compared with HH+DMSO group, the viability was significantly increased, the levels of LDH and TNF-α and apoptotic rate were significantly decreased, the protein levels of Bax, TLR4 and p-IκB were significantly decreased, the protein level of Bcl-2 was significantly increased in HH+emodin groups (P<0.05). Over-expression of TLR4 reversed the effect of emodin on promoting the viability and inhibiting apoptosis in the BV2 cells. CONCLUSION: Emodin has a protective effect on hypoglycemia/hypoxia induced microglia, and its mechanism may be related to the inactivation of TLR4/NF-κB signaling pathway.     

Omega-3 Fatty Acid Intervention Suppresses Lipopolysaccharide-Induced Inflammation and Weight Loss in Mice

Bacterial endotoxin lipopolysaccharide (LPS)-induced sepsis is a critical medical condition, characterized by a severe systemic inflammation and rapid loss of muscle mass. Preventive and therapeutic strategies for this complex disease are still lacking. Here, we evaluated the effect of omega-3 (n-3) polyunsaturated fatty acid (PUFA) intervention on LPS-challenged mice with respect to inflammation, body weight and the expression of Toll-like receptor 4 (TLR4) pathway components. LPS administration induced a dramatic loss of body weight within two days. Treatment with n-3 PUFA not only stopped loss of body weight but also gradually reversed it back to baseline levels within one week. Accordingly, the animals treated with n-3 PUFA exhibited markedly lower levels of inflammatory cytokines or markers in plasma and tissues, as well as down-regulation of TLR4 pathway components compared to animals without n-3 PUFA treatment or those treated with omega-6 PUFA. Our data demonstrate that n-3 PUFA intervention can suppress LPS-induced inflammation and weight loss via, at least in part, down-regulation of pro-inflammatory targets of the TLR4 signaling pathway, and highlight the therapeutic potential of n-3 PUFA in the management of sepsis.     

赤眼鳟Toll样受体9基因的cDNA全长克隆及表达特性分析

为探究赤眼鳟(Squaliobarbus curriculus)Toll样受体9基因(toll–like receptor 9,ScTLR9)的结构特性及其参与免疫应答草鱼呼肠弧病毒(grass carp reovirus,GCRV)感染时的表达特性,采用RACE技术克隆Sc TLR9基因c DNA全长序列,采用实时荧光定量PCR技术比较分析ScTLR9免疫应答GCRV的表达特性。结果显示:ScTLR9的cDNA全长为3 687 bp,编码1 059个氨基酸,有17个富含亮氨酸重复序列(leucine rich repeats,LRRs)结构域。ScTLR9在被检测的9个组织中均有表达,在中肾和头肾中的表达量最高;感染GCRV后,基因TLR9在赤眼鳟和草鱼头肾中的表达量均上调,在赤眼鳟头肾中的表达量于感染后12 h达到峰值,推测TLR9参与了赤眼鳟抗GCRV的免疫应答,且在抗GCRV入侵免疫反应中赤眼鳟较草鱼的免疫应答更为迅速。     

Wip1正调控TLR4信号介导Ⅰ型干扰素的表达

Wip1是由基因ppm1d编码的丝氨酸/苏氨酸磷酸酶,不仅在细胞应激反应、增殖分化中起重要作用,还参与调控炎症反应和肿瘤发生等过程。Wip1能抑制TNF-α诱导的NF-κB激活,而NF-κB又是TLR4信号通路中的重要组分,因此推测Wip1在LPS诱导的TLR4信号通路中可能起调控作用。Western印记结果显示,在Wip1基因敲除的小鼠胚胎成纤维细胞(MEFs,Mouse embryonic fibroblasts)中,TLR4信号诱导的IRF3磷酸化程度显著降低。与此相一致,实时荧光定量PCR实验揭示,受LPS刺激后,Wip1基因敲除的MEFs比野生型细胞产生更少的I型干扰素IFN-β和干扰素诱导基因(Interferon-stimulated genes,ISGs),如Mx1,Mx2和Rasd2。将野生型和磷酸酶失活的Wip1分别回补进Wip1缺失的MEFs,构建了野生型和突变体Wip1回补的细胞系进一步验证该结果。通过实时荧光定量PCR研究,发现野生型和磷酸酶失活的Wip1均能促进LPS诱导的ISGs的表达,Wip1不依赖于其磷酸酶活性来促进TLR4介导的I型干扰素反应。研究首次发现Wip1能通过增强IRF3的磷酸化来促进I型干扰素的表达,揭示了机体抵御病毒感染的新机制。