Innate Immunity

先天免疫

• 批准号: 7592594
• 负责人: David M. Segal
• 金额: $ 108.05万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592594
• 关键词: Affect; Affinity; Antibodies; Antigen Receptors; Asparagine; Binding; Binding Sites; Buffers; C-terminal; Cell Degranulation; Cell surface; Cells; Collaborations; Complex; Cytolysis; Cytoplasmic Tail; DNA; Decompression Sickness; Development; Double-Stranded RNA; Early Endosome; Endocytosis; Equilibrium; Extracellular Domain; Face; Generations; Genes; Glycolipids; Histamine; Histamine Receptor; Histidine; Human; Immune response; Immune system; Immunization; Infection; Inflammatory Response; Investigation; Laboratories; Lateral; Ligand Binding; Ligands; Location; Mediating; Molecular; Molecular Profiling; Molecular Structure; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Natural Immunity; Nature; Nucleic Acids; Numbers; Oligonucleotides; Pattern; Pattern recognition receptor; Phenotype; Play; Polysaccharides; Process; Proteins; Resolution; Role; Shapes; Signal Transduction; Site; Solutions; Source; Structure; Surface; System; T-Lymphocyte; TLR3 gene; Testing; Toll-Like Receptor 1; Toll-like receptors; Transgenic Organisms; Vesicle; acquired immunity; base; beta pleated sheet; cell type; cytokine; dimer; human TLR3 protein; in vivo; interest; mast cell; microorganism; mouse model; paralogous gene; pathogen; receptor; receptor function; receptor structure function; response; size

The vertebrate immune response to infection begins with the recognition by the innate immune system of conserved molecular signatures of pathogens, known as PAMPs (Pathogen Associated Molecular Patterns), provoking an immediate and often massive inflammatory response. The innate response holds the pathogen in check, but also plays a crucial role in the generation of acquired immunity. The recognition of PAMPs by the innate system is mediated by a number of receptors, of which the Toll-like Receptors (TLRs) play a prominent role. Unlike the antigen receptors of acquired immunity, the TLRs are encoded by a limited number of germline genes, ten in humans; however, in spite of their small numbers, the TLRs recognize a remarkably wide variety of PAMPs including glycolipids, proteins, and nucleic acids. We have been investigating several aspects of TLR structure and function: 1. The molecular basis for the recognition of a wide array of PAMPs by TLRs is a main interest of my laboratory. In collaboration with Dr. David Davies (LMB, NIDDK), we have recently succeeded in expressing, crystallizing, and determining the molecular structure of the TLR3 extracellular domain (ECD). The structure consists of a solenoid of 23 turns, bent into a horseshoe shape, with a large beta-sheet on the concave surface. The molecules is heavily glycosylated, except that one lateral face of the horseshoe is totally devoid of glycan. Although we have not yet obtained a high resolution structure of a TLR3-ligand (dsRNA) complex, we have located the dsRNA binding site by mutational analysis. The ligand binds on the glycan-free lateral face of the TLR3 molecule to histidine and asparagine residues, near the C-terminal end . We have now completed an investigation of the interaction of dsRNA oligos with TLR3-ECD protein. We have found that purified TLR3-ECD binds dsRNA specifically via a defined ligand-binding site and with an affinity that increases with buffer acidification and ligand size. TLR3-ECD is monomeric in solution, but it forms dimers when bound to dsRNA. These dimers are stabilized by cooperative interactions between the two TLR3-ECDs in a pair, and multiple TLR3ecd dimers bind to long dsRNAs. The smallest oligonucleotides that form stable complexes with TLR3ecd (40-50 bp) each bind one TLR3ecd dimer, and these are also the smallest dsRNAs that activate TLR3 in cells. Thus, we have demonstrated that the TLR3 signaling unit is a ligand-bound dimer of TLR3 molecules. Looking past TLR3, we plan to express and examine ECDs from other TLR paralogs, to see how they differ in structure and ligand binding function from TLR3. 2. Nucleic acid PAMPs such as dsRNA, ssRNA, and CpG DNA, ligands for TLRs 3, 7, 8, and 9, are normally sequestered within microorganisms and become available to interact with TLRs only after the pathogen is endocytosed and lysed intracellularly. By contrast, TLRs 1, 2, 4, 5, 6, and 10 interact with PAMPs that are normally present in the medium, and these TLRs are, as expected, located on the cell surface. Therefore, correct cellular localization is essential for TLR function. We have studied the localization of TLR9, and motifs within the TLR9 molecule that mediate this localization. We have found that TLR9 is located, prior to stimulation, in the ER, and that it interacts with CpG DNA in early endosomes. Both the cytoplasmic and extracellular domains contain internalization signals, and we have also located, within the cytoplasmic domain of TLR9 two regions that control intracellular localization. We are now initiating studies on the intracellular location of TLR3. In the case of TLR3, we have already established that the activation signal varies depending upon the cell type, and we are testing the hypothesis that signaling is influenced by intracellular localization, in particular the pH of the intracellular vesicle that contains TLR3. 3. TLRs play a pivotal role in acquired immunity by triggering the maturation of DC to competent APC, capable of priming naive T cells. Based on our observation that DC also express histamine receptors, we hypothesized that histamine would have an effect on the maturation process. In testing this hypothesis we found that histamine profoundly alters the cytokines released by DC during TLR induced maturation, and as a result, histamine exposure causes DC to polarize naive T cells toward a Th2 phenotype. Mast cells are the major source of histamine, and they are often located in close proximity to DC. We therefore hypothesized that mast cell degranulation at a site of immunization would alter the nature of the immune response, by acting on neighboring DC. By using a mouse model with adoptively transferred transgenic T cells, we have demonstrated an effect of mast cell degranulation on T cell polarization in vivo. By using mice that are deficient in mast cells, we have now shown that mast cells affect the Th1/Th2 balance in mice, and we are now asking whether this has an effect upon the type of antibody produced

脊椎动物对感染的免疫反应始于先天免疫系统对病原体保守分子特征的识别，称为PAMPs（病原体相关分子模式），引发立即且通常是大量的炎症反应。先天反应控制病原体，但在获得性免疫的产生中也起着至关重要的作用。先天系统对PAMPs的识别是由许多受体介导的，其中toll样受体（TLRs）起着突出的作用。与获得性免疫的抗原受体不同，tlr由有限数量的种系基因编码，在人类中有10个；然而，尽管它们的数量很少，但tlr识别的pamp种类非常广泛，包括糖脂、蛋白质和核酸。我们从几个方面研究了TLR的结构和功能：1。tlr识别一系列PAMPs的分子基础是我实验室的主要兴趣。通过与David Davies博士（LMB， NIDDK）的合作，我们最近成功地表达、结晶并确定了TLR3细胞外结构域（ECD）的分子结构。该结构由一个23圈的螺线管组成，弯曲成马蹄形，凹面上有一个大的β片。分子糖基化程度很高，除了马蹄铁的侧面完全没有糖基化。虽然我们还没有获得tlr3 -配体（dsRNA）复合物的高分辨率结构，但我们已经通过突变分析确定了dsRNA的结合位点。该配体结合在TLR3分子无聚糖的侧面，靠近c末端的组氨酸和天冬酰胺残基。我们现在已经完成了dsRNA寡核苷酸与TLR3-ECD蛋白相互作用的研究。我们发现纯化的TLR3-ECD通过一个确定的配体结合位点特异性地与dsRNA结合，并且随着缓冲酸化和配体大小的增加而增加亲和力。TLR3-ECD在溶液中为单体，但与dsRNA结合时形成二聚体。这些二聚体通过一对tlr3 - ecd之间的合作相互作用而稳定，并且多个TLR3ecd二聚体结合到长dsrna上。与TLR3ecd形成稳定复合物的最小寡核苷酸（40-50 bp）每个结合一个TLR3ecd二聚体，这些也是激活细胞中TLR3的最小dsRNAs。因此，我们已经证明了TLR3信号单元是TLR3分子的配体结合二聚体。展望过去的TLR3，我们计划表达和检查来自其他TLR类似物的ECDs，看看它们在结构和配体结合功能上与TLR3有何不同。2. 核酸PAMPs，如dsRNA、ssRNA和CpG DNA， TLRs 3、7、8和9的配体，通常被隔离在微生物内，只有在病原体被内噬和细胞内裂解后才能与TLRs相互作用。相比之下，TLRs 1、2、4、5、6和10与通常存在于介质中的pamp相互作用，并且这些TLRs如预期的那样位于细胞表面。因此，正确的细胞定位对TLR的功能至关重要。我们研究了TLR9的定位，以及TLR9分子中介导这种定位的基序。我们发现TLR9在刺激之前位于内质网中，并与早期内体中的CpG DNA相互作用。细胞质和细胞外结构域都包含内化信号，我们还在TLR9的细胞质结构域中发现了两个控制细胞内定位的区域。我们目前正在启动TLR3细胞内定位的研究。在TLR3的情况下，我们已经确定激活信号取决于细胞类型，我们正在测试信号传导受细胞内定位影响的假设，特别是包含TLR3的细胞内囊泡的pH值。3. tlr在获得性免疫中发挥关键作用，通过触发DC成熟为能够启动幼稚T细胞的APC。根据我们的观察，DC也表达组胺受体，我们假设组胺可能对成熟过程有影响。在验证这一假设时，我们发现组胺深刻地改变了DC在TLR诱导的成熟过程中释放的细胞因子，因此，组胺暴露导致DC将幼稚T细胞极化为Th2表型。肥大细胞是组胺的主要来源，它们通常位于DC附近。因此，我们假设免疫部位的肥大细胞脱颗粒会通过作用于邻近的DC而改变免疫反应的性质。通过采用过继转移转基因T细胞的小鼠模型，我们证明了肥大细胞脱颗粒对体内T细胞极化的影响。通过使用肥大细胞缺乏的小鼠，我们现在已经证明肥大细胞影响小鼠的Th1/Th2平衡，我们现在想知道这是否对产生的抗体类型有影响