Immunological basis of M13 phage vaccine: Regulation under MyD88 and TLR9 signaling

Peptide-displaying bacteriophages induce mimotope-specific antibody responses, suggesting a novel application of phage-display library as bacteriophage vaccine. We examined the antibody response against M13 phage in mice induced by an i.p. administration of M13 phage in phosphate-buffered saline. We showed here that firstly, mice showed strong IgG antibody responses, particularly, in IgG2b, IgG2c, and IgG3 subclasses even in primary responses. Secondly, IgG production in primary response is totally dependent on MyD88 signaling. These responses were almost comparable, but slightly weaker, in TLR2-, TLR4- and TLR7-deficient mice relative to wild-type mice, suggesting that this enhancing effect is not due to plausible LPS contamination. Thirdly, although primary IgG1 response was not detected in wild-type mice, remarkable IgG1 response was induced in TLR9-deficient mice, suggesting that TLR9 pathway functions as regulatory, but not a simple augmenting signaling cascade, and furthermore, the enhanced IgG1 response was not due to adjuvant effect of single-stranded DNA derived from M13 phage. Thus, innate immunity including TLR regulation is crucial for M13 phage vaccine design.     

Characterization of bbtTICAM from amphioxus suggests the emergence of a MyD88-independent pathway in basal chordates

The MyD88-independent pathway, one of the two crucial TLR signaling routes, is thought to be a vertebrate innovation. However, a novel Toll/interleukin-1 receptor (TIR) adaptor, designated bbtTICAM, which was identified in the basal chordate amphioxus, links this pathway to invertebrates. The protein architecture of bbtTICAM is similar to that of vertebrate TICAM1 (TIR-containing adaptor molecule-1, also known as TRIF), while phylogenetic analysis based on the TIR domain indicated that bbtTICAM is the oldest ortholog of vertebrate TICAM1 and TICAM2 (TIR-containing adaptor molecule-2, also known as TRAM). Similar to human TICAM1, bbtTICAM activates NF-κB in a MyD88-independent manner by interacting with receptor interacting protein (RIP) via its RHIM motif. Such activation requires bbtTICAM to form homodimers in endosomes, and it may be negatively regulated by amphioxus SARM (sterile α and armadillo motif-containing protein) and TRAF2. However, bbtTICAM did not induce the production of type I interferon. Thus, our study not only presents the ancestral features of vertebrate TICAM1 and TICAM2, but also reveals the evolutionary origin of the MyD88-independent pathway from basal chordates, which will aid in understanding the development of the vertebrate TLR network.     

水牛MyD88cDNA的克隆与原核表达

采用RT-PCR方法从水牛外周血白细胞总RNA中扩增出髓样分化因子88 (mydoid differentiation factor 88,MyD88) cDNA序列,PCR产物分离纯化后,与pMD20-T载体连接,重组质粒经PCR、酶切鉴定后测序,并进行生物信息学分析;构建pET28a-MyD88表达载体,并将其转化至E.coli BL21 (DE3),经IPTG诱导表达后,进行SDS-PAGE、镍柱亲和层析纯化和Western blotting分析.结果显示,克隆到的水牛MyD88 cDNA全长为1 189 bp,含有1个891 bp的开放阅读框,编码296个氨基酸,理论等电点为5.65.经IPTG诱导表达后,得到一个带His·Tag的约39 kD的重组融合蛋白.用抗His单克隆抗体进行Western blotting,得到1条约39 kD特异性抗体结合带,表明水牛MyD88原核表达载体成功构建并表达.本研究为进一步开展水牛MyD88的结构功能分析奠定了基础.     

Regulation of MyD88 Aggregation and the MyD88-dependent Signaling Pathway by Sequestosome 1 and Histone Deacetylase 6

MyD88 is an essential adaptor molecule for Toll-like receptors (TLRs) and interleukin (IL)-1 receptor. MyD88 is thought to be present as condensed forms or aggregated structures in the cytoplasm, although the reason has not yet been clear. Here, we show that endogenous MyD88 is present as small speckle-like condensed structures, formation of which depends on MyD88 dimerization. In addition, formation of large aggregated structures is related to cytoplasmic accumulation of sequestosome 1 (SQSTM1; also known as p62) and histone deacetylase 6 (HDAC6), which are involved in accumulation of polyubiquitinated proteins. A gene knockdown study revealed that SQSTM1 and HDAC6 were required for MyD88 aggregation and exhibited a suppressive effect on TLR ligand-induced expression of IL-6 and NOS2 in RAW264.7 cells. SQSTM1 and HDAC6 were partially involved in suppression of several TLR4-mediated signaling events, including activation of p38 and JNK, but they hardly affected degradation of IκBα (inhibitor of nuclear factor κB). Biochemical induction of MyD88 oligomerization induced recruitment of SQSTM1 and HDAC6 to the MyD88-TRAF6 signaling complex. Repression of SQSTM1 and HDAC6 enhanced formation of the MyD88-TRAF6 complex and conversely decreased interaction of the ubiquitin-specific negative regulator CYLD with the complex. Furthermore, ubiquitin-binding regions on SQSTM1 and HDAC6 were essential for MyD88 aggregation but were not required for interaction with the MyD88 complex. Thus, our study reveals not only that SQSTM1 and HDAC6 are important determinants of aggregated localization of MyD88 but also that MyD88 activates a machinery of polyubiquitinated protein accumulation that has a modulatory effect on MyD88-dependent signal transduction.     

松江鲈(Trachiderm usfasciatus)髓样分化因子MyD88基因克隆与组织表达分析

髓样分化因子(My D88)是TOLL样受体介导的信号通路中的一个关键接头分子,通过激活核转录因子(nuclear factor-kappa B,NF-κB)而参与机体的先天免疫。克隆了松江鲈的My D88基因(命名为Tf My D88),并对该基因进行了生物信息学和表达模式分析。结果显示,Tf My D88 c DNA序列全长1 555 bp,5'UTR长89 bp,3'UTR长599 bp;开放阅读框长度为867 bp,编码288个氨基酸。SMART软件预测Tf My D88分子的N端为一个保守的死亡结构域(death domain,DD),C端存在典型的TIR(Toll/interleukin-1 receptor)结构域。Tf My D88与其它脊椎动物My D88的氨基酸相似性达57.58%-82.64%,系统进化树分析表明Tf My D88与同属鲈形总目的花鲈和鳜鱼聚在一起,所有鱼类My D88聚为一支。Real-time PCR检测显示Tf My D88广泛表达于松江鲈各组织,但在鳃中的相对表达量最高;其次为脾脏和皮肤。LPS(lipopolysaccharide)刺激后,Tf My D88在松江鲈的血液、肝脏、皮肤、脾脏均出现明显上调。刺激2 h后,在血液Tf My D88表达量升高了近60倍,在皮肤中的表达量也升高了27倍。上述结果表明Tf My D88可能参与松江鲈先天免疫。     

Toll-like receptors and fungal infections: the role of TLR2, TLR4 and MyD88 in paracoccidioidomycosis

The aim of this minireview is to present a concise view of the most important pattern recognition receptors used by the innate immune system to sense and control pathogen growth into host tissues. A brief review of the role of Toll-like receptors (TLRs) in fungal infections followed by some recent results on the function of TLR4, TLR2 and the MyD88 adaptor molecule in the pathogenesis of paracoccidioidomycosis are presented.     

Atg5 regulates formation of MyD88 condensed structures and MyD88-dependent signal transduction

MyD88 is known as an essential adaptor protein for Toll-like receptors (TLRs). Previous studies have shown that transfected MyD88 forms condensed structures in the cytoplasm. However, upon TLR stimulation, there is little formation of endogenous MyD88 condensed structures. Thus, the formation of MyD88 condensed structures is tightly suppressed, but the mechanism and significance of this suppression are currently unknown. Here we show that Atg5, a key regulatory protein of autophagy, inhibits the formation of MyD88 condensed structures. We found that endogenous MyD88 had already formed condensed structures in Atg5-deficient cells and that the formation of condensed structures was further enhanced by TLR stimulation. This suppressive effect of Atg5 may not be associated with autophagic processes because MyD88 itself was not degraded and because TLR stimulation did not induce LC3 punctate formation and LC3 conversion. Immunoprecipitation analysis revealed that Atg5 could interact with MyD88. Furthermore, Atg5 deficiency increased formation of the MyD88–TRAF6 signaling complex induced by TLR stimulation, and it enhanced activation of NF-κB signaling but not MAPKs and Akt. These findings indicate that Atg5 regulates the formation of MyD88 condensed structures through association with MyD88 and eventually exerts a modulatory effect on MyD88-dependent signaling.     

鸡MyD88-TIR的同源模建

髓样分化因子(MyD88)是Toll受体(TLR)信号通路中的一个关键接头分子,在传递信息和介导炎症反应中具有重要的作用。对鸡MyD88(Myeloid differentiation primary response protein MyD88)的TIR(Toll-interleukin1-resistance)区域进行同源建模,并评估其可用性,为进一步研究MyD88与TLR(Toll receptor)相互作用的原理奠定基础。通过结构域分析、模板相似性搜索和序列比对、初始建模、精修和动力学优化,立体化学结构和能量合理性评估,获得未知三维结构的鸡MyD88-TIR三维模型。结果表明,鸡MyD88包含DEATH和TIR两个结构域,所模拟的MyD88-TIR三维模型二面角构象和氨基酸能量分布以及主侧链立体化学特性合理。     

MyD88 in myofibroblasts enhances colitis-associated tumorigenesis via promoting macrophage M2 polarization

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MyD88抑制乙型肝炎病毒复制依赖活化NF-κB信号通路

目的 研究髓样细胞分化蛋白(MyD88)抗乙型肝炎病毒(HBV)效应的作用机制。方法 构建MyD88的截短突变体,获得核因子kappa B(NF-κB)超抑制剂IkBa-SR或者NF-κB信号通路激活剂IKKα/IKKβ的表达质粒,分别与HBV复制型质粒瞬时转染Huh7细胞,检测细胞上清液中HBeAg,HBsAg的表达以及胞质中HBV复制中间体DNA的含量,并以NF-κB依赖的荧光素酶报道系统检测它们活化NF-κB的程度。结果 MyD88全长蛋白和2个截短突变体M(1-151)、M(151-296)活化NF-κB的程度与其抑制HBV蛋白以及复制中间体DNA合成的能力相一致。与空载相比,表达NF-κB信号通路激活剂IKKα/IKKβ的质粒共同瞬转细胞后,转染MyD88和HBV表达质粒的细胞中NF-κB的通路明显活化,同时HBV core蛋白的合成显著降低;而NF-κB的超抑制剂IκBα-SR共同瞬转的细胞中core蛋白的表达量显著增加,检测细胞培养上清液中HBeAg和HBsAg及胞质中HBV复制中间体DNA的合成,得到相似结果。结论 NF-κB信号通路的活化在MyD88抑制HBV复制中发挥了关键作用     

Mycobacterial infection in MyD88-deficient mice

MyD88 is an adaptor protein that plays a major role in TLR/IL-1 receptor family signaling. To understand the role of MyD88 in the development of murine tuberculosis in vivo, MyD88 knockout (KO) mice aerially were infected with Mycobacterium tuberculosis. Infected MyD88 mice were not highly susceptible to M. tuberculosis infection, but they developed granulomatous pulmonary lesions with neutrophil infiltration which were larger than those in wild-type (WT) mice (P < 0.01). The pulmonary tissue levels of mRNA for iNOS and IL-18 were slightly lower, but levels of mRNA for IL-1 beta, IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TGF-beta were higher in MyD88 KO mice. IFN-gamma, TNF-alpha, IL-1 beta, and IL-12 also were high in the sera of MyD88 KO mice. There were no statistically significant differences in the expression of TNF-alpha, IL-12, and ICAM-1 mRNA between MyD88 KO and WT mice. Thus, MyD88 deficiency did not influence the development of murine tuberculosis. NF-kappa B activity was similar in the alveolar macrophages from the lung tissues of MyD88 KO and WT mice. Also, there may be a TLR2-specific, MyD88-independent IL-1 receptor/TLR-mediated pathway to activate NF-kappa B in the host defense against mycobacterial infection.     

猪髓样分化因子MyD88特异性shRNA干扰载体的构建筛选及干扰效果评价

髓样分化因子My D88(myeloid differentiation factor 88)信号通路是一个具有多种调节功能的传导通路,在免疫反应、炎症反应及肿瘤的发生和发展过程中均发挥重要作用。构建猪(Sus scrofa)My D88基因的shRNA干扰载体,并在转录水平和蛋白质表达水平对其干扰效果进行验证,以筛选出干扰效果最优的干扰载体。根据猪My D88基因(Gen Bank登录号:KC766424.1)全长c DNA序列,利用Invitrogen公司在线设计软件设计出4对shRNA干扰序列,退火成双链后,分别将其插入到p Yr-1.1载体中,构建My D88基因的shRNA真核表达载体p Yr-1.1-pig My D88-sh1、p Yr-1.1-pig My D88-sh2、p Yr-1.1-pig My D88-sh3、p Yr-1.1-pig My D88-sh4,并通过双酶切和测序对其进行鉴定。构建成功后转染猪肺泡巨噬细胞3D4/2,通过Real-time PCR及Western blot验证My D88基因的表达水平,以及对LPS刺激后炎症因子TNF-α基因表达水平的影响。结果表明,所构建的猪My D88基因的特异性shRNA表达载体均可显著降低猪My D88 mRNA和蛋白质的表达水平(P0.05),干扰效率分别达到36%、67%、60%、69%;相比于未干扰组,LPS刺激My D88沉默之后的巨噬细胞,炎症因子TNF-α基因表达水平显著下降(P0.05),表明所构建猪My D88干扰载体干扰效果较好。     

MyD88在抗衣原体感染的病理损伤过程中的作用

<正>沙眼衣原体(Chlamydia trachomatis,Ct)是引起泌尿生殖道感染的常见性病病原体之一,并可导致不孕、异位妊娠、宫颈鳞状细胞癌等并发症[1]。但有关沙眼衣原体确切的致病机制及机体的抗感染机制目前尚不清     

赤眼鳟MyD88基因cDNA克隆与表达特性研究

实验使用RACE-PCR技术获得了赤眼鳟(Squaliobarbus curriculus)髓样分化因子88 (Myeloid differentiationfactor 88, MyD88)的cDNA全长, 命名为ScMyD88。ScMyD88的cDNA全长为1779 bp, 其中开放阅读框855 bp, 共编码284个氨基酸残基, 推导的蛋白质分子量为33.053 kD, 理论等电点为5.66。赤眼鳟MyD88具有典型的MyD88结构特征, 包括死亡结构域和TIR结构域(Toll-IL-1 receptor domain, TIR), 其氨基酸序列和鲤科鱼类具有高度保守性, 相似性达到了90%以上, 特别是和武昌鱼相比, 相似性达到了98%。在检测的9个赤眼鳟组织和器官中均有MyD88表达, 其中肝脏、头肾和体肾中表达水平最高, 在脑中表达量最低。在草鱼呼肠弧病毒(Grass carp reovirus, GCRV)感染初期(12h内), MyD88在赤眼鳟免疫组织中表达水平急剧上升, 特别是在脾脏和体肾中尤为明显, 随后恢复正常水平。研究表明, MyD88在赤眼鳟抵抗GCRV入侵的免疫应答反应初期发挥了重要作用。     

以MyD88 TIR二聚化为靶点的抗炎抑制剂筛选模型的建立

MyD88是IL-1R/TLR受体超家族向细胞内转导胞外信号时募集到受体胞浆尾部的重要接头蛋白．由TIR结构域介导的MyD88分子同源二聚化是它招募到受体胞浆尾部的前提,然后二聚化的MyD88再募集下游信号分子,传递信号,引发促炎基因的表达．本研究旨在建立一种模型,以实现活细胞原位的、基于荧光信号变化的MyD88二聚化抑制物的高通量筛选．我们分别构建了MyD88 TIR与GFP和RFP的融合蛋白表达质粒,瞬时转染HeLa细胞,在488 nm激发光下,转染GFP-MyD88 TIR和RFP-MyD88 TIR细胞,检测到绿色荧光与红色荧光间的共振能量转移(FRET)．而当细胞转染GFP-MyD88 TIR和RFP或RFP-MyD88 TIR和GFP,因TIR二聚化不能实现,FRET效率受到严重影响．实验结果提示,依赖双阳性表达GFP-MyD88 TIR和RFP-MyD88 TIR的细胞株,检测不同化合物对于荧光FRET效率的影响,可以建立MyD88 TIR二聚化抑制药物的筛选模型．此外,我们构建了原核表达质粒,利用纯化的His-MyD88 TIR分别与GST或GST-MyD88 TIR蛋白进行体外结合实验,发现GST-MyD88 TIR(而非GST)可以与His-MyD88 TIR相互结合．结果的差异性提示,利用His-MyD88 TIR和GST-MyD88 TIR体外结合实验分析,可以进一步确定抑制物是否直接阻断了TIR的相互作用．结合真核细胞的荧光FRET阻断结果和原核表达的重组蛋白相互作用分析,可确定MyD88 TIR二聚化的抑制物．利用这一模型可以对商品化的小分子库、自行制备的天然产物组分进行广泛的筛选,从中获得有效抑制MyD88二聚化的化合物,参与对MyD88信号通路依赖的慢性炎症、自身免疫性疾病的药物治疗．     

Effects of myeloid differentiation primary response gene 88 (MyD88) activation on Helicobacter infection in vivo and induction of a Th17 response

Background:  Helicobacter pylori is a spiral‐shaped Gram‐negative microaerophilic bacterium associated with a number of gastrointestinal disorders, including gastritis, peptic ulcers, and gastric cancer. Several studies have implicated a Th17 response as a key to protective immunity against Helicobacter. Materials and Methods:  Wild type (WT) and MyD88‐deficient (MyD88^?/?) mice in the C57BL/6 background were infected with H. felis for 6 and 25 weeks and colonization density and host response evaluated. Real‐time PCR was used to determine the expression of cytokines and antimicrobial peptides in the gastric tissue of mice. Results:  mRNA expression levels of the Th17 cytokines interleukin‐17A (IL‐17A) and IL‐22 were markedly up‐regulated in WT compared with MyD88^?/? mice both at 6 and at 25 weeks in response to infection with H. felis, indicating that induction of Th17 responses depends on MyD88 signaling. Furthermore, reduction in the expression of Th17‐dependent intestinal antimicrobial peptide lipocalin‐2 was linked with increased bacterial burden in the absence of MyD88 signaling. Conclusion:  We provide evidence showing that MyD88‐dependent signaling is required for the host to induce a Th17 response for the control of Helicobacter infection.     

Lys694Arg polymorphism leads to blunted responses to LPS by interfering TLR4 with recruitment of MyD88

TLR4 polymorphisms such as Asp299Gly and Thr399Ile related to Gram-negative sepsis have been reported to result in significantly blunted responsiveness to LPS. Our study group previously screened other TLR4 polymorphic variants by checking the NF-κB activation in comparison to wild type (WT) TLR4 in human embryonic kidney 293T cells. In this study, we found that the Lys694Arg (K694R) polymorphism reduced the activation of NF-κB, and the production of downstream inflammatory factors IL-1, TNF-α and IL-6, representing the K694R polymorphism, led to blunted responsiveness to LPS. Then, we examined the influence of the K694R polymorphism on total and cell-surface TLR4 expression by Western blotting and flow cytometry, respectively, but observed no differences between the K694R polymorphism and WT TLR4. We also used co-immunoprecipitation to determine the interaction of the K694R polymorphism and WT TLR4 with their co-receptor myeloid differentiation factor 2 (MD2) and their downstream signal adaptor MyD88. We found that K694R reduced the recruitment of MyD88 in TLR4 signalling but had no impact on the interaction with MD2.