细菌类脂A结构与功能研究进展

类脂A分布在革兰氏阴性细菌的外表层,它是脂多糖分子的疏水基团.脂多糖,俗名内毒素,可以引起致命的脓毒症、内毒素血症和多器官功能障碍综合症等疾病.近年来的研究发现:脂多糖分子中只有类脂A部分具有内毒素的活性.当细菌侵入人体后,其表面的类脂A可以刺激宿主细胞表面的Toll样受体-4,在细胞内引起一连串的反应,产生一系列的细胞因子.本文根据近年来内毒素领域的国际研究进展,系统综述了类脂A的结构特征、合成途径和致病机理,并在此基础上分析了内毒素在疫苗开发领域的应用前景.     

急性出血坏死性胰腺炎肝损伤中TLR2, 4mRNA和蛋白的表达

目的:观察急性出血坏死性胰腺炎肝损伤中TLR-2、TLR4的表达水平,分析TLR2和TLR4在AHNP肝损伤中的表达意义。方法:48只成年Wistar大鼠作为实验动物,随机分为对照组和造模组各24只,造模组利用牛磺胆酸钠建立AHNP模型,在造模后3 h、12 h以及24 h时,每组分别各取8只大鼠,应用RT-PCR法检测TLR2、TLR4mRNA表达水平,应用Western blot检测肝脏组织中TLR2、TLR4蛋白表达水平。结果:造模后,造模组TLR2mRNA、TLR4mRNA、TLR2蛋白、TLR4蛋白显著升高,且在造模后12 h出现峰值,与同时段对照组相比差异显著(P0.01)。结论:急性出血坏死性胰腺炎肝损伤组织中TLR2、TLR4mRNA和蛋白表达水平异常升高,TLR2、TLR4可能参与了急性出血坏死性胰腺炎肝损伤发生发展过程。     

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.     

髓样分化蛋白-2在识别和转导内毒素信号中的作用

脂多糖(LPS)通过TLR4介导细胞炎症反应.研究表明,髓样分化蛋白-2(MD-2)通过与TLR4形成复合物参与LPS诱导的细胞信号过程.TLR4/MD-2复合物中的MD-2结合LPS后,引起TLR4低聚化,进而激发下游信号.MD-2合成后,大部分在内质网/高尔基体和TLR4结合,然后以TLR4/MD-2复合物的形式在细胞表面表达.这既能调节TLR4的胞内分布,又能辅助TLR4识别LPS.还有一部分MD-2释放到血浆中,形成可溶性的MD-2(sMD-2).sMD-2在CD14参与下,能结合血浆中的LPS,形成LPS-sMD-2复合物从而辅助只表达TLR4而不表达MD-2的细胞识别LPS,但过度表达的sMD-2又能抑制LPS信号.MD-2在TLR4介导的内毒素识别和信号转导过程中发挥了重要的调控作用.     

Molecular dynamics study of phospholipase A2 on a membrane surface

The desolvation of lipid molecules in a complex of the enzyme human synovial phospholipase A₂ with a lipid membrane is investigated as a mechanism that enhances the overall activity of the enzyme. For this purpose the interaction of the enzyme phospholipase A₂ with a dilauryl-phosphatityl-ethanolamin (DLPE) membrane monolayer surface has been studied by means of molecular dynamics simulations. Two enzyme-membrane complexes, a loose and a tight complex, are considered. For comparison, simulations are also carried out for the enzyme in aqueous solution. The conformation, dynamics, and energetics of the three systems are compared, and the interactions between the protein and lipid molecules are analyzed. Free energies of solvation are calculated for the lipid molecules in the enzyme–membrane interface. Along with the calculated dielectric susceptibility at this interface, the results show the desolvation of lipids in a tightly bound, but not in a loosely bound protein-membrane complex. The desolvated lipids are found to interact mainly with hydrophobic protein residues, Including Leu-2, Val-3, Ala-18, Leu-19, Phe-24, Val-31, and Phe-70. The results also explain why the turnover rate of phospholipase A₂ complexed to a membrane is enhanced after a critical amount of negatively charged reaction product is accumulated. © 1996 Wiley-Liss, Inc.     

TLR4 mutation and HSP60-induced cell death in adult mouse cardiac myocytes

Extracellular (ex) HSP60 is increasingly recognized as an agent of cell injury. Previously, we reported that low endotoxin exHSP60 causes cardiac myocyte apoptosis. Our findings supported a role for Toll-like receptor (TLR) 4 in HSP60 mediated apoptosis. To further investigate the involvement of TLR4 in cardiac injury, we studied adult cardiac myocytes from C3H/HeJ (HeJ) mice, which have a mutant, nonfunctional TLR4, and compared the results with parallel studies using wild-type (WT) mice. Nuclear factor κB (NFκB) activation is an early step downstream of TLR4. NFκB was activated 1 h after treatment with HSP60 in WT, but not HeJ mouse myocytes. ExHSP60 caused apoptosis in cardiac myocytes from WT mice, but not in myocytes from the HeJ mutants. To further elucidate the importance of exHSP60 in cardiac myocyte injury, both WT and HeJ mutant isolated mouse adult cardiac myocytes were exposed to hypoxia/reoxygenation. Anti-HSP60 antibody treatment reduced apoptosis in the WT group, but had no effect on the HeJ mutant myocytes. Unexpectedly, necrosis was also decreased in the HeJ mutants. Necrosis after hypoxia/reoxygenation in WT cardiac myocytes was mediated in part by TLR2 and TLR4 through rapid activation of PKCα, followed by increased expression of Nox2, and this was ameliorated by blocking antibodies to TLR2/4. These studies provide further evidence that TLR4 mediates exHSP60-associated apoptosis and that exHSP60 has an important role in cardiac myocyte injury, both apoptotic and necrotic.     

Ligation of TLR2 and TLR4 on murine bone marrow-derived mesenchymal stem cells triggers differential effects on their immunosuppressive activity

Mesenchymal stem cells (MSCs) have potent regulatory effects on immune and inflammatory responses. Recently the findings of functional TLR expression on MSC implicates these receptors in the function established for MSCs. Here we specially investigated the effects of TLR2, 4 ligation in mice MSC on migration, modulation of allogeneic mixed lymphocytes reaction (allo-MLR) and inducing Treg cells. We demonstrated that ligation of TLR2, but not TLR4, could significantly inhibit migration of MSC, impair MSC-mediated immunosuppression on allo-MLR, and reduce MSC-mediated expansion of CD4+CD25+Foxp3+ regulatory T cells. Compared with TLR4 activated MSCs and non-TLR activated MSC, TLR2 activation induced a relatively lower level of CXCL-10 mRNA and protein expressions which has been elucidated to act in concert with other soluble factor in MSC-mediated immunomodulation. These data indicate that TLR2 and TLR4 ligation had different effects on immunomodulatory capability of murine BMSCs, which should be considered in their use for treating inflammatory diseases.     

Limited contribution of Toll-like receptor 2 and 4 to the host response to a fungal infectious pathogen, Cryptococcus neoformans

The present study was designed to elucidate the role of Toll-like receptor (TLR) 2 and TLR4 in the host response to Cryptococcus neoformans. Both TLR2 knockout (KO) and TLR4KO mice produced interleukin-1beta (IL-1beta), IL-6, IL-12p40 and tumor necrosis factor-alpha (TNF-alpha) in sera and cleared this fungal pathogen from infected lungs at a comparable level to control littermate (LM) mice. Synthesis of these cytokines was not significantly different in the lungs of these KO mice and LM mice, although IL-1beta, IL-6 and IL-12p40 tended to be lower in TLR2KO, but not TLR4KO, mice than in controls. In addition, there was no significant reduction detected in the synthesis of IL-12 and TNF-alpha by bone marrow-derived dendritic cells from TLR2KO and TLR4KO mice upon stimulation with live yeast cells. Finally, HEK293 cells expressing either TLR2/dectin-1 or TLR4/MD2/CD14 did not respond to C. neoformans in the activation of nuclear factor kappa B (NFkappaB) detected by a luciferase assay. Our results suggest that TLR2 and TLR4 do not or only marginally contribute to the host and cellular response to this pathogen.     

Molecular dynamics simulation of interleukin-2 and its complex and determination of the binding free energy

Interleukin-2 (IL-2) protein belongs to the signal modulator cytokine's family and therefore it is prevalent for immunological responses. It has been identified as a centrally important potential drug target for the inhibition of protein-protein interactions; so as to suppress the immunological responses associated with autoimmune, inflammatory and immunological diseases, and cancer. In the present work, we have performed two independent 100?ns of molecular dynamics (MD) simulations on the apo IL-2 protein and its ligand-bound complex (with a potent inhibitor FRG), to study the effect of inhibitor binding on the dynamics and stability of the protein. The calculation of binding free energy via post-processing end state method of Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) and Molecular Mechanics Generalised Born Surface Area (MM-GBSA) has inferred a good correlation in accordance with the already reported experimental data, demonstrating that the free energy of binding calculated by the two methods has no significant difference. The investigation of individual components of free energy revealed that the association of IL-2 protein with FRG ligand is primarily driven by the van der Waals energy contribution that represents the non-polar/hydrophobic energy contribution as dominant in this case of ligand binding.     

利用荧光共振能量转移技术研究活细胞TLR4与MD-2作用结构域

脂多糖(LPS)的识别和信号转导是宿主发生防御反应的关键,Toll样受体4(TLR4)与髓样分化蛋白-2(MD-2)形成复合物在LPS的识别及其信号转导中发挥了重要作用．研究TLR4与MD-2结合的功能结构域,对于深入了解LPS信号转导机制及其内毒素休克的防治具有重要意义．运用基于强度的三通道荧光共振能量转移技术(FRET)及基因突变和转染技术,研究了活细胞TLR4与MD-2作用的结构域．结果表明：N端Glu²⁴～Met⁴¹缺失使TLR4与MD-2结合能力明显下降;LPS刺激后TLR4聚合迅速增加,而缺失Glu²⁴～Met⁴¹的TLR4不能聚合．上述结果提示,TLR4的Glu²⁴～Met⁴¹不仅是结合MD-2的区域,并且还参与了LPS刺激后TLR4的聚合作用．     

Toll样受体4与病毒感染的研究进展

Toll样受体4（TLR4）是固有免疫系统中能够识别病原相关分子模式的受体家族成员,可识别革兰氏阴性菌的脂多糖（LPS）而在细菌感染性疾病的发生中起重要作用。近年来越来越多的研究发现,TLR4还广泛参与病毒感染性疾病的发生和病毒的免疫逃逸,由于其信号转导通路的独特性和细胞定位的可变性,再次引起人们极大的研究兴趣。该文将介绍TLR4的生物学特性、信号转导通路及TLR4与病毒感染的最新研究进展。