Regulation of spontaneous intestinal tumorigenesis through the adaptor protein MyD88

Inflammation is increasingly recognized as an important component of tumorigenesis, although the mechanisms and pathways involved are not well understood. Tumor development is regulated by products of several modifier genes, but instructions for their tumor-specific expression are currently unknown. We show that the signaling through the adaptor protein MyD88 has a critical role in spontaneous tumor development in mice with heterozygous mutation in the adenomatous polyposis coli (APC) gene. We found that MyD88-dependent signaling controls the expression of several key modifier genes of intestinal tumorigenesis and has a critical role in both spontaneous and carcinogen-induced tumor development. This study thus reveals the important role of an innate immune signaling pathway in intestinal tumorigenesis.     

Adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling

Innate immune signals mediated by Toll-like receptors (TLRs) have been thought to contribute considerably to the antibody-enhancing effects of vaccine adjuvants. However, we report here that mice deficient in the critical signaling components for TLR mount robust antibody responses to T cell-dependent antigen given in four typical adjuvants: alum, Freund's complete adjuvant, Freund's incomplete adjuvant, and monophosphoryl-lipid A/trehalose dicorynomycolate adjuvant. We conclude that TLR signaling does not account for the action of classical adjuvants and does not fully explain the action of a strong adjuvant containing a TLR ligand. This may have important implications in the use and development of vaccine adjuvants.     

Cathepsin K-dependent toll-like receptor 9 signaling revealed in experimental arthritis

Cathepsin K was originally identified as an osteoclast-specific lysosomal protease, the inhibitor of which has been considered might have therapeutic potential. We show that inhibition of cathepsin K could potently suppress autoimmune inflammation of the joints as well as osteoclastic bone resorption in autoimmune arthritis. Furthermore, cathepsin K-/- mice were resistant to experimental autoimmune encephalomyelitis. Pharmacological inhibition or targeted disruption of cathepsin K resulted in defective Toll-like receptor 9 signaling in dendritic cells in response to unmethylated CpG DNA, which in turn led to attenuated induction of T helper 17 cells, without affecting the antigen-presenting ability of dendritic cells. These results suggest that cathepsin K plays an important role in the immune system and may serve as a valid therapeutic target in autoimmune diseases.     

Structural basis of toll-like receptor 3 signaling with double-stranded RNA

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA), a molecular signature of most viruses, and triggers inflammatory responses that prevent viral spread. TLR3 ectodomains (ECDs) dimerize on oligonucleotides of at least 40 to 50 base pairs in length, the minimal length required for signal transduction. To establish the molecular basis for ligand binding and signaling, we determined the crystal structure of a complex between two mouse TLR3-ECDs and dsRNA at 3.4 angstrom resolution. Each TLR3-ECD binds dsRNA at two sites located at opposite ends of the TLR3 horseshoe, and an intermolecular contact between the two TLR3-ECD C-terminal domains coordinates and stabilizes the dimer. This juxtaposition could mediate downstream signaling by dimerizing the cytoplasmic Toll interleukin-1 receptor (TIR) domains. The overall shape of the TLR3-ECD does not change upon binding to dsRNA.     

Pyogenic bacterial infections in humans with MyD88 deficiency

MyD88 is a key downstream adapter for most Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 deficiency in mice leads to susceptibility to a broad range of pathogens in experimental settings of infection. We describe a distinct situation in a natural setting of human infection. Nine children with autosomal recessive MyD88 deficiency suffered from life-threatening, often recurrent pyogenic bacterial infections, including invasive pneumococcal disease. However, these patients were otherwise healthy, with normal resistance to other microbes. Their clinical status improved with age, but not due to any cellular leakiness in MyD88 deficiency. The MyD88-dependent TLRs and IL-1Rs are therefore essential for protective immunity to a small number of pyogenic bacteria, but redundant for host defense to most natural infections.     

多子小瓜虫感染后草鱼TLR信号通路基因 的表达动态

[目的]明确草鱼Toll样受体(Toll-like receptor,TLR)信号通路基因在防御多子小瓜虫(Ichthyophthirius multifiliis)感染过程中的免疫作用,为有效防控鱼类多子小瓜虫感染提供理论参考.[方法]以多子小瓜虫感染草鱼后,采用实时荧光定量PCR检测分析在不同时间点(感染后6 h、12 h、1 d、2 d、3 d、5 d和7 d)草鱼部分TLRs基因(TLR1、TLR2、TLR4、TLR9、TLR20和TLR21)、接头蛋白基因(MyD88和TRIF)、信号转导分子(IRAK4和IRAK1)及细胞因子(IL-1β、TNF-α和IFN)在其皮肤和脾脏中的表达动态变化.[结果]草鱼感染多子小瓜虫后,TLR1、TLR2、TLR9、TLR20和TLR21基因在皮肤和脾脏中的表达主要呈上调趋势,TLR4基因在皮肤中主要呈上调表达,在脾脏中则主要呈下调表达;TLR信号通路接头蛋白基因MyD88和TRIF的表达变化趋势明显不同,其中,MyD88基因的表达在感染后第1~3 d均呈显著上调趋势(P<0.05,下同),而TRIF基因的表达在整个试验过程中绝大多数时间点与对照组无显著差异(P>0.05);IRAK4和IRAK1在皮肤和脾脏中的表达也主要呈上调趋势,但在脾脏中IRAK4在感染后第1 d和IRAK1在感染后第6 h的表达呈显著下调趋势;IL-1β和TNF-α在绝大多数时间点均显著上调,但IFN的表达基本没有变化.[结论]草鱼TLR信号通路中的部分TLRs基因(TL1、TLR2、TLR4、TLR9、TLR20和TLR21)、接头蛋白基因(MyD88)、信号转导分子(IRAK4和IRAK1)及下游细胞因子(IL-1β和TNF-α)均参与防御多子小瓜虫感染的免疫反应,尤其是在感染早期和中期发挥关键作用.     

鸡源复合益生菌对青年白羽肉杂鸡免疫球蛋白和Toll样受体通路的影响

为研究鸡源复合益生菌对青年白羽肉杂鸡血清免疫球蛋白和肠道Toll样受体通路的影响,将180只健康的28日龄白羽肉杂鸡随机分为A(基础饲粮)、B(基础饲粮中添加10¹¹ CFU·kg^-1益生菌)、C(基础饲粮中添加2×10¹¹ CFU·kg^-1益生菌)3个处理,每个处理3个重复,每个重复20只鸡。试验期21 d。结果表明:与A组相比,B组和C组鸡血清中IgG、IgM水平显著(P<0.05)升高,B组和C组鸡肠道TLR2、TLR4、Myd88、TRAF-6、AP-1蛋白表达及mRNA相对表达量显著(P<0.05)升高。与B组相比,C组鸡血清IgG、IgM、IgA水平均无明显差异(P>0.05),鸡肠道TLR4、Myd88蛋白表达显著(P<0.05)升高,Myd88基因mRNA相对表达量显著(P<0.05)升高。综上,添加不同浓度的鸡源复合益生菌能够提高白羽肉杂鸡血清中IgG、IgM水平,通过Toll样受体通路增强肉鸡的免疫功能。     

A distinct signaling pathway used by the IgG-containing B cell antigen receptor

The immunoglobulin G (IgG)-containing B lymphocyte antigen receptor (IgG-BCR) transmits a signal distinct from that of IgM-BCR or IgD-BCR, although all three use the same signal-transducing component, Igalpha/Igbeta. Here we demonstrate that the inhibitory coreceptor CD22 down-modulates signaling through IgM-BCR and IgD-BCR, but not that through IgG-BCR, because of the IgG cytoplasmic tail, which prevents CD22 phosphorylation. These results suggest that the cytoplasmic tail of IgG specifically enhances IgG-BCR signaling by preventing CD22-mediated signal inhibition. Enhanced signaling through IgG-BCR may be involved in efficient IgG production, which is crucial for immunity to pathogens.     

Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production

Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men. Similar gender disparity is seen in mice given a chemical carcinogen, diethylnitrosamine (DEN). DEN administration caused greater increases in serum interleukin-6 (IL-6) concentration in males than it did in females. Furthermore, ablation of IL-6 abolished the gender differences in hepatocarcinogenesis in mice. DEN exposure promoted production of IL-6 in Kupffer cells (KCs) in a manner dependent on the Toll-like receptor adaptor protein MyD88, ablation of which also protected male mice from DEN-induced hepatocarcinogenesis. Estrogen inhibited secretion of IL-6 from KCs exposed to necrotic hepatocytes and reduced circulating concentrations of IL-6 in DEN-treated male mice. We propose that estrogen-mediated inhibition of IL-6 production by KCs reduces liver cancer risk in females, and these findings may be used to prevent HCC in males.     

Illumination of the melanopsin signaling pathway

In mammals, a small population of intrinsically photosensitive retinal ganglion cells (ipRGCs) plays a key role in the regulation of nonvisual photic responses, such as behavioral responses to light, pineal melatonin synthesis, pupillary light reflex, and sleep latency. These ipRGCs also express melanopsin (Opn4), a putative opsin-family photopigment that has been shown to play a role in mediating these nonvisual photic responses. Melanopsin is required for the function of this inner retinal pathway, but its precise role in generating photic responses has not yet been determined. We found that expression of melanopsin in Xenopus oocytes results in light-dependent activation of membrane currents through the Galpha(q)/Galpha(11) G protein pathway, with an action spectrum closely matching that of melanopsin-expressing ipRGCs and of behavioral responses to light in mice lacking rods and cones. When coexpressed with arrestins, melanopsin could use all-trans-retinaldehyde as a chromophore, which suggests that it may function as a bireactive opsin. We also found that melanopsin could activate the cation channel TRPC3, a mammalian homolog of the Drosophila phototransduction channels TRP and TRPL. Melanopsin therefore signals more like an invertebrate opsin than like a classical vertebrate rod-and-cone opsin.     

Autophagy and the nutritional signaling pathway

During their growth and development, animals adapt to tremendous changes in order to survive. These include responses to both environmental and physiological changes and autophagy is one of most important adaptive and regulatory mechanisms. Autophagy is defined as an autolytic process to clear damaged cellular organelles and recycle the nutrients via lysosomic degradation. The process of autophagy responds to special conditions such as nutrient withdrawal. Once autophagy is induced, phagophores form and then elongate and curve to form autophagosomes. Autophagosomes then engulf cargo, fuse with endosomes, and finally fuse with lysosomes for maturation. During the initiation process, the ATG1/ULK1 (unc-51-like kinase 1) and VPS34 (which encodes a class III phosphatidylinositol (PtdIns) 3-kinase) complexes are critical in recruitment and assembly of other complexes required for autophagy. The process of autophagy is regulated by autophagy related genes (ATGs). Amino acid and energy starvation mediate autophagy by activating mTORC1 (mammalian target of rapamycin) and AMP-activated protein kinase (AMPK). AMPK is the energy status sensor, the core nutrient signaling component and the metabolic kinase of cells. This review mainly focuses on the mechanism of autophagy regulated by nutrient signaling especially for the two important complexes, ULK1 and VPS34.     

MyD88抑制剂ST2825对丝状支原体山羊亚种 感染宿主细胞的影响

为分析MyD88在丝状支原体山羊亚种(Mmc)感染肺泡上皮细胞中的分子机制。以山羊肺泡上皮细胞为材料,用不同浓度的MyD88抑制剂(ST2825)与细胞孵育,MTT法检测细胞活性,试剂盒检测caspase-3的活性,以及实时定量PCR检测MyD88和TNF-a mRNA的表达水平,试剂盒检测H_2O_2浓度和NO浓度。当ST2825浓度为5、10和20μmol·L~(-1)不影响细胞活性,而浓度为40μmol·L~(-1)时细胞活性显著降低;感染比例(MOI)为10,感染时间为8、12和24 h,Mmc可以显著提高MyD88 mRNA的表达水平(P0.01);可显著提高caspase-3的活性,而ST2825能够显著降低caspase-3(P0.05);感染时间为24 h,Mmc可显著提高TNF-a mRNA的表达水平、H_2O_2浓度和NO浓度(P0.05),而ST2825在10和20μmol·L~(-1)的浓度时,可显著降低Mmc介导的caspase-3活性、H_2O_2浓度以及LDH水平(P0.05),ST2825在5、10和20μmol·L~(-1)浓度时可显著降低TNF-a mRNA的表达水平和NO浓度(P0.05)。     

黄芪多糖对犬血清中白介素水平及肠道Toll样受体信号通路的影响

【目的】研究黄芪多糖(APS)对犬血清中白介素(IL)水平及肠道Toll样受体信号通路的影响,探索APS发挥免疫调节效应的作用及机制。【方法】将18只比格犬平均分为对照组(基础日粮)、APS低剂量组(基础日粮+质量分数1%的黄芪多糖)和APS高剂量组(基础日粮+质量分数2%的黄芪多糖),饲喂2周。于试验0,7,14 d采集血清,ELISA法检测血清中IL-2、IL-4和IL-6水平;第14天,解剖全部犬,采集十二指肠,用RT-qPCR、免疫组化(IHC)和Western Blots等技术检测TLR4信号转导途径相关蛋白(TLR4、TLR7、MyD88、TRAF-6和AP-1)的表达。【结果】试验7和14 d时,与对照组相比,APS低剂量组犬血清中IL-2、IL-4和IL-6水平显著(P0.05)或极显著(P0.01)升高,APS高剂量组犬血清中IL-2、IL-4和IL-6水平均极显著升高(P0.01)。与对照组相比,APS低剂量组犬肠道组织中TLR4、TLR7、MyD88、TRAF-6和AP-1蛋白表达光密度值、蛋白及其mRNA相对表达量显著升高(P0.05),APS高剂量组显著(P0.05)或极显著(P0.01)升高,APS低剂量组与高剂量组之间差异不显著(P0.05)。【结论】黄芪多糖可显著提高犬血清中IL-2、IL-4和IL-6水平,并通过调节Toll样受体通路蛋白表达增强犬机体免疫力。     

凡纳滨对虾Toll样受体基因cDNA片段的克隆及序列分析

对凡纳滨对虾(Litopenaeus vannamei)Toll样受体基因的cDNA片段进行了克隆.从凡纳滨对虾提取肌肉、鳃和肝胰腺中的总RNA,根据黑腹果蝇(Drosophila melanogaster)Toll样受体的TIR区设计引物,从鳃中逆转录扩增得到cDNA序列,进行测序.所得序列结果与其他物种的已知TIR及TLR氨基酸序列进行聚类分析建立系统进化树,并进行氨基酸序列同源性比较.结果表明所得序列与斑节对虾(Penaeus monodon)、赤拟谷盗(Tribolium castaneum)、果蝇(Drosophila melanogaster)、意大利蜜蜂(Apis mellifera)、线虫(Caenorhabditiselegans)、紫海胆(Strongylocentrotus purpuratus)的Toll样基因TIR区域的氨基酸序列有较高的相似性.     

Functional genomic analysis of the Wnt-wingless signaling pathway

The Wnt-Wingless (Wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Aberrant Wnt signaling has been linked to human disease. In the present study, we used a genomewide RNA interference (RNAi) screen in Drosophila cells to screen for regulators of the Wnt pathway. We identified 238 potential regulators, which include known pathway components, genes with functions not previously linked to this pathway, and genes with no previously assigned functions. Reciprocal-Best-Blast analyses reveal that 50% of the genes identified in the screen have human orthologs, of which approximately 18% are associated with human disease. Functional assays of selected genes from the cell-based screen in Drosophila, mammalian cells, and zebrafish embryos demonstrated that these genes have evolutionarily conserved functions in Wnt signaling. High-throughput RNAi screens in cultured cells, followed by functional analyses in model organisms, prove to be a rapid means of identifying regulators of signaling pathways implicated in development and disease.     

凡纳滨对虾Toll样受体基因cDNA片段的克隆及序列分析

对凡纳滨对虾(Litopenaeus vannamei)Toll样受体基因的cDNA片段进行了克隆.从凡纳滨对虾提取肌肉、鳃和肝胰腺中的总RNA,根据黑腹果蝇(Drosophila melanogaster)Toll样受体的TIR区设计引物,从鳃中逆转录扩增得到cDNA序列,进行测序.所得序列结果与其他物种的已知TIR及TLR氨基酸序列进行聚类分析建立系统进化树,并进行氨基酸序列同源性比较.结果表明所得序列与斑节对虾(Penaeus monodon)、赤拟谷盗(Tribolium castaneum)、果蝇(Drosophila melanogaster)、意大利蜜蜂(Apis mellifera)、线虫(Caenorhabditiselegans)、紫海胆(Strongylocentrotus purpuratus)的Toll样基因TIR区域的氨基酸序列有较高的相似性.     

Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8

Double-stranded ribonucleic acid (dsRNA) serves as a danger signal associated with viral infection and leads to stimulation of innate immune cells. In contrast, the immunostimulatory potential of single-stranded RNA (ssRNA) is poorly understood and innate immune receptors for ssRNA are unknown. We report that guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from human immunodeficiency virus-1 (HIV-1) stimulate dendritic cells (DC) and macrophages to secrete interferon-alpha and proinflammatory, as well as regulatory, cytokines. By using Toll-like receptor (TLR)-deficient mice and genetic complementation, we show that murine TLR7 and human TLR8 mediate species-specific recognition of GU-rich ssRNA. These data suggest that ssRNA represents a physiological ligand for TLR7 and TLR8.