草鱼呼肠孤病毒上调稀有鮈鲫鳃中TLR3和Mx基因的表达

鳃暴露在水环境中,增加了对疾病的易感性。为了研究稀有鮈鲫人工感染草鱼呼肠孤病毒过程中鳃部先天性免疫反应机制,我们克隆了抗病毒效应分子Mx基因的部分序列,用适时荧光定量PCR检测双链RNA的模式识别受体（Toll-like receptor3,TLR3）及I型干扰素指示基因Mx的表达。TLR3和Mx基因的表达在注射病毒后12h显著升高（p〈0.05）,TLR3的表达水平在注射后48h恢复到正常水平（p〉0.05）,而Mx的高水平表达一直持续到实验结束（p〈0.05）。结果表明在GCRV感染中,鳃能发生局部免疫反应,其干扰素途径被激活。     

小鼠Toll样受体3基因的克隆及真核表达

采用RT-PCR方法从小鼠巨噬细胞中克隆小鼠Toll样受体3(TLR3)基因,基因测序表明获得了小鼠全长TLR3cDNA,构建了真核表达质粒p3XFLAG-CMV-7.1-TLR3.重组质粒转染293T细胞,Western blotting检测蛋白表达,表达蛋白质的相对分子量与预计相符.采用TLR3的阳性刺激物poly(I∶C)刺激重组质粒转染的293T细胞,双荧光素酶报告基因系统检测发现能激活下游转录因子NF-κB的转录活性,并能诱导TLR3下游细胞因子IL-6和TNF-α的表达.小鼠TLR3基因的克隆和表达,为研究TLR3介导的信号通路及其在抗病毒免疫中的作用打下基础.     

赤眼鳟Mx基因全长cDNA克隆及其经GCRV攻毒后的组织表达分析

为研究赤眼鳟(Squaliobarbus curriculus)Mx蛋白(Myxovirus resistance protein)的功能, 采用简并PCR和SMART RACE方法从赤眼鳟脾脏中克隆得到Mx基因全长cDNA, 并通过生物信息学方法分析其同源性, 再利用实时荧光定量PCR (RT-qPCR)检测其在脾、肝、肠、肾等9个组织中的表达, 以及感染草鱼呼肠孤病毒(Reovirus of Grass carp) GCRV-104后不同时间点赤眼鳟Mx的时空表达规律。结果表明: 赤眼鳟Mx基因cDNA序列(ScMx)全长2325 bp, 包含5'-UTR 40 bp, 3'-UTR 371 bp和ORF 1884 bp, 共编码627个氨基酸, 其编码的Mx蛋白分子量约为70.9 kD, 理论等电点 pI 为 8.25, 具有脊椎动物Mx蛋白共有的结构特征; 赤眼鳟Mx与鲫鱼Mx3同源性最高; Mx在赤眼鳟脾、肝、肠、肾等9个组织中均有表达, 其中肝脏中的相对表达量最高, 脾脏次之, 肠组织中的表达量最低; 经GCRV-104病毒感染刺激后, ScMx在肝和脾组织中的表达量显著上调, 均在48h到达峰值, 分别为对照组的10倍(肝)和5倍(脾), 且在这两个组织中的表达模式相似, 均表现为先升高后下降的波动型变化趋势。研究表明ScMx参与了赤眼鳟抗GCRV-104病毒的免疫反应。       

MicroRNA调控固有免疫应答的分子机制

MicroRNA（miRNA）是近几年继siRNA之后非编码RNA研究的又一热点．它通过与靶mRNA的特异性结合,在转录后水平上对基因表达进行调控．研究表明,miRNA可能参与脊椎动物固有免疫应答的多个环节．在病原微生物感染时,它们不仅成为重要的固有免疫受体活化后的信号调节分子,而且能够直接干扰病毒复制而发挥抗病毒效应．miRNA可能与经典的固有免疫应答体系共同组成机体抵御病原微生物入侵的“第一道防线”．同时,病原微生物,特别是病毒还可以通过自己编码miRNA或者改变宿主细胞miRNA表达谱直接或间接地干扰很多宿主免疫相关基因的表达,实现逃逸机体免疫清除的目的．因此,miRNA水平的相瓦作用可能是病原微生物与其宿丰展开免疫“博弈”的重要战场．     

TLR2, TLR3, and TLR4 activation specifically alters the oxidative status of intestinal epithelial cells

Intestinal inflammatory diseases are the result of multiple processes, including mucosal oxidative stress and perturbed homeostasis between commensal bacteria and mucosal immunity. Toll-like receptors (TLRs) recognize molecular-associated microorganisms' patterns and trigger innate immunity responses contributing to intestinal homeostasis and inflammatory responses. However, TLRs effects on redox balance in intestinal mucosa remain unknown. Therefore, the present study analyzes the effect of TLR2, TLR3, and TLR4 on both oxidative damage of lipids and proteins, and the activity of antioxidant enzymes in enterocyte-like Caco-2 cells. The results show that the activation of these TLRs increased lipid and protein oxidation levels; however, the effect on the antioxidant enzymes activity is different depending on the TLR activated. These results suggest that the activation of TLR2, TLR3, and TLR4 might affect intestinal inflammation by not only their inherent innate immunity responses, but also their pro-oxidative effects on intestinal epithelial cells.     

Toll-like Receptors 3, 4, and 7 Are Expressed in the Enteric Nervous System and Dorsal Root Ganglia

The aim of the present study was to evaluate the expression of innate immunity receptors belonging to the Toll-like family in the neural plexuses of the different tracts of murine intestine, of the human ileum, and in lower dorsal root ganglia (DRGs) from where extrinsic afferents to these plexuses originate. Results obtained by immunohistochemistry and immunofluorescence on paraffin-embedded tissue and whole-mount preparations show that Toll-like receptors (TLRs) -3 and -7, recognizing viral RNA, and TLR4, recognizing lipopolysaccharide (membrane component of Gram-negative bacteria), are expressed in the myenteric and submucous plexuses of murine intestine and human ileum, and in DRGs primary sensory neurons. They also show that TLR4 immunostaining is stronger in murine distal large bowel. In murine tissue, expression of TLRs was present in both neurons and glial cells. These observations indicate that the enteric neural network might be directly activated by bacterial and viral components and is therefore more in the forefront than previously envisaged in defense responses of the intestinal wall and in the cross-talk with intestinal microbiota. They also highlight the presence of a peripheral neural network that by way of hardwired neurotransmission could potentially convey to the central nervous system specific information on our microbial counterpart and invading or potentially invading pathogens. (J Histochem Cytochem 57:1013–1023, 2009)     

Characterization of equine and other vertebrate TLR3, TLR7, and TLR8 genes

Toll-like receptors 3, 7, and 8 (TLR3, TLR7, and TLR8) were studied in the genomes of the domestic horse and several other mammals. The messenger RNA sequences and exon/intron structures of these TLR genes were determined. An equine bacterial artificial chromosome clone containing the TLR3 gene was assigned by fluorescent in situ hybridization to the horse chromosomal location ECA27q16–q17 and this map location was confirmed using an equine radiation hybrid panel. Direct sequencing revealed 13 single-nucleotide polymorphisms in the coding regions of the equine TLR 3, 7, and 8 genes. Of these polymorphisms, 12 were not previously reported. The allelic frequency was estimated for each single-nucleotide polymorphism from genotyping data obtained for 154 animals from five horse breeds. Some of these frequencies varied significantly among different horse breeds. Domain architecture predictions for the three equine TLR protein sequences revealed several conserved regions within the variable leucine-rich repeats between the corresponding horse and cattle TLR proteins. A phylogenetic analysis did not indicate that any significant exchanges had occurred between paralogous TLR7 and TLR8 genes in 20 vertebrate species analyzed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mycobacterial infection in TLR2 and TLR6 knockout mice

To investigate the role of TLR in the development of murine tuberculosis in vivo, TLR2 and TLR6 knockout (KO) mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne infection apparatus. Both TLR2 and TLR6 KO mice survived until sacrifice at 12 weeks after infection. Infected TLR2 KO mice developed granulomatous pulmonary lesions with neutrophil infiltration, which were slightly larger in size than those in wild-type mice. Pulmonary levels of the mRNAs for inducible nitric oxide synthase (iNOS), TNF-alpha, TGF-beta, IL-1beta, and IL-2 were significantly lower, but levels of the mRNAs for IL-4 and IL-6 were higher, than in wild-type (WT) mice. No significant difference was recognized in cytokine mRNA expression between TLR2 KO and WT mice at 12 weeks after infection. DNA binding by NF-kappaB was low in TLR2 KO mice. On the other hand, TLR6 KO mice were not different from WT mice in terms of pulmonary histopathology, mRNA expression and CFU assay. Therefore, TLR2 does not play an essential role in the pathogenesis of murine tuberculosis, although it is important for defense against mycobacterial infection.     

猪Mx1和牛Mx1蛋白在PK-15细胞中的表达及其对伪狂犬病病毒的抑制

目的:研究猪Mx1和牛Mx1蛋白在PK-15细胞中的表达并检测其是否对伪狂犬病病毒(PRV)具有抑制作用。方法:从IBRS-2细胞和MDBK细胞中分别调取猪Mx1和牛Mx1基因,并克隆到pc DNA3.1/myc-His(-)B,构建得到真核重组表达质粒,以脂质体转染的方法将其分别导入到PK-15细胞,从mRNA水平和蛋白质水平鉴定重组质粒在细胞内的表达情况,然后用细胞毒性试剂盒检测这两种蛋白是否对PK-15细胞具有毒性。之后,通过荧光定量PCR检测猪Mx1和牛Mx1在攻毒后不同时间、不同攻毒剂量的条件下对PRV的抑制情况,并观察100TCID50病毒攻击细胞72h后的病变程度。结果:成功克隆了猪Mx1和牛Mx1基因,经mRNA水平和蛋白质水平证实,两种重组质粒在PK-15细胞内能够正常表达。从荧光定量PCR和细胞病变的角度来看,细胞内表达的Mx1蛋白对PRV具有显著性的抑制(P0.001)。结论:猪Mx1和牛Mx1基因在PK-15细胞中表达的Mx1蛋白能够抑制PRV在胞内的复制。     

Down modulation of human TLR3 function by a monoclonal antibody

Toll-like receptors are a family of pattern-recognition receptors that contribute to the innate immune response. Toll-like receptor 3 (TLR3) signals in response to foreign, endogenous and synthetic ligands including viral dsRNA, bacterial RNA, mitochondrial RNA, endogenous necrotic cell mRNA and the synthetic dsRNA analog, poly(I:C). We have generated a monoclonal antibody (mAb CNTO2424) that recognizes the extracellular domain (ECD) of human TLR3 in a conformation-dependent manner. CNTO2424 down-regulates poly(I:C)-induced production of IL-6, IL-8, MCP-1, RANTES, and IP-10 in human lung epithelial cells. In addition, mAb CNTO2424 was able to interfere with the known TLR3-dependent signaling pathways, namely NF-κB, IRF-3/ISRE, and p38 MAPK. The generation of this neutralizing anti-TLR3 mAb provides a unique tool to better understand TLR3 signaling and potential cross-talk between TLR3 and other molecules.     

The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

Recognition of viral dsRNA by Toll-like receptor 3 (TLR3) leads to induction of interferons (IFNs) and proinflammatory cytokines, and innate antiviral response. Here we identified the RNA-binding protein Mex3B as a positive regulator of TLR3-mediated signaling by expression cloning screens. Cells from Mex3b^−/− mice exhibited reduced production of IFN-β in response to the dsRNA analog poly(I:C) but not infection with RNA viruses. Mex3b^−/− mice injected with poly(I:C) was more resistant to poly(I:C)-induced death. Mex3B was associated with TLR3 in the endosomes. It bound to dsRNA and increased the dsRNA-binding activity of TLR3. Mex3B also promoted the proteolytic processing of TLR3, which is critical for its activation. Mutants of Mex3B lacking its RNA-binding activity inhibited TLR3-mediated IFN-β induction. These findings suggest that Mex3B acts as a coreceptor of TLR3 in innate antiviral response.     

猪Mx1基因第14外显子多态性分析及新突变位点的 发现

采用PCR-RFLP方法对国内外7个猪种Mx1基因第14外显子的多态性进行分析, 共检测到3个等位基因, 6种基因型。其中杜洛克中仅存在AA基因型, 苏太猪中存在全部基因型, 只有在梅山猪和具有梅山猪血统的苏太猪中出现基因型BB。所有猪种中, 只有在地方猪种和培育猪种中出现等位基因B, 所有猪种除松辽黑猪外均以A为优势等位基因。卡方检验结果表明, 不同猪种间基因型分布差异较大, 梅山猪和松辽黑猪与其他所有猪种的基因型频率差异极显著(P＜0.01) , 苏太猪与除皮特兰猪外的所有猪种的基因型频率差异也极显著(P＜0.01) , 淮猪与杜洛克和约克夏这两个国外猪种基因型频率差异不显著(P＞0.05), 而与皮特兰和其他地方猪种的基因型频率均存在极显著差异(P＜0.01) 。通过测序在扩增片段中新发现了3种类型的碱基突变, 前2个分别导致了Thr和Glu向Ala和Arg的替换, 最后一个突变不引起氨基酸的变化, 且后两个突变位点为BB基因型所特有。     

Molecular Characterization of Mx1 Gene in Native Indian Breeds of Chicken

The genetic polymorphism of Mx1 gene was explored in Indian chicken breeds. PCR-RFLP analysis in 102?bp fragment of partial intron 13 and partial exon 14 of Mx1 gene revealed two genotypes viz. RS and SS with two alleles viz. R and S both in Naked Neck and Tellicherry breeds of chicken. The homozygous genotype RR was not identified. When deduced amino acid sequences were compared, the asparagine amino acid was found to be substituted in “R” allele for serine in “S” allele. PCR-SSCP analysis of 284?bp fragment in 5′-UTR and partial promoter region revealed three genotypes viz. CC, CG, and CH with three different alleles viz. C, G, and H in Naked Neck breed of chicken and five genotypes viz. DI, JK, KK, KL, and KM with six different alleles viz. D, I, J, K, L, and M in Tellicherry breed of chicken. The homozygous genotypes viz. GG and HH in Naked Neck and DD, II, JJ, LL, and MM in Tellicherry chicken was not identified. The nucleotide substitution rate estimated to be in the range of 0.004–0.011. The identified genetic variation can be helpful for better insight to disease resistance property of the Mx1 gene.     

Differential COX-2 induction by viral and bacterial PAMPs: Consequences for cytokine and interferon responses and implications for anti-viral COX-2 directed therapies

Cyclooxygenase 2 (COX)-2 is induced by bacterial and viral infections and has complex, poorly understood roles in anti-pathogen immunity. Here, we use a knock-in luciferase reporter model to image Cox2 expression across a range of tissues in mice following treatment with the either the prototypical bacterial pathogen-associated molecular pattern (PAMP), LPS, which activates Toll-like receptor (TLR)4, or with poly(I:C), a viral PAMP, which activates TLR3. LPS induced Cox2 expression in all tissues examined. In contrast, poly(I:C) elicited a milder response, limited to a subset of tissues. A panel of cytokines and interferons was measured in plasma of wild-type, Cox1^−/− and Cox2^−/− mice treated with LPS, poly(I:C), MALP2 (TLR2/6), Pam3CSK4 (TLR2/1), R-848 (TLR7/8) or CpG ODN (TLR9), to establish whether/how each COX isoform modulates specific PAMP/TLR responses. Only LPS induced notable loss of condition in mice (inactivity, hunching, piloerection). However, all TLR agonists produced cytokine responses, many of which were modulated in specific fashions by Cox1 or Cox2 gene deletion. Notably we observed opposing effects of Cox2 gene deletion on the responses to the bacterial PAMP, LPS, and the viral PAMP, poly(I:C), consistent with the differing abilities of the PAMPs to induce Cox2 expression. Cox2 gene deletion limited the plasma IL-1β and interferon-γ responses and hypothermia produced by LPS. In contrast, in response to poly(I:C), Cox2^−/− mice exhibited enhanced plasma interferon (IFNα,β,γ,λ) and related cytokine responses (IP-10, IL-12). These observations suggest that a COX-2 selective inhibitor, given early in infection, may enhance and/or prolong endogenous interferon responses, and thereby increase anti-viral immunity.     

Toll-like receptors control autophagy

Autophagy is a newly recognized innate defense mechanism, acting as a cell-autonomous system for elimination of intracellular pathogens. The signals and signalling pathways inducing autophagy in response to pathogen invasion are presently not known. Here we show that autophagy is controlled by recognizing conserved pathogen-associated molecular patterns (PAMPs). We screened a PAMP library for effects on autophagy in RAW 264.7 macrophages and found that several prototype Toll-like receptor (TLR) ligands induced autophagy. Single-stranded RNA and TLR7 generated the most potent effects. Induction of autophagy via TLR7 depended on MyD88 expression. Stimulation of autophagy with TLR7 ligands was functional in eliminating intracellular microbes, even when the target pathogen was normally not associated with TLR7 signalling. These findings link two innate immunity defense systems, TLR signalling and autophagy, provide a potential molecular mechanism for induction of autophagy in response to pathogen invasion, and show that the newly recognized ability of TLR ligands to stimulate autophagy can be used to treat intracellular pathogens.