Innate Immunity

脊椎动物对感染的免疫反应始于天然免疫系统对病原体保守的分子特征的识别，称为PAMPs(病原体相关分子模式)，引发立即且往往是大规模的炎症反应。与生俱来的反应抑制了病原体，但也在后天免疫的产生中发挥了关键作用。先天系统对PAMPs的识别是由多种受体介导的，其中Toll样受体(TLRs)起着重要的作用。与获得性免疫的抗原受体不同，TLRs由有限数量的生殖系基因编码，在人类中为10个；然而，尽管数量很少，TLRs识别的PAMP种类非常广泛，包括糖脂、蛋白质和核酸。我们一直在研究TLR的结构和功能的几个方面：1.TLRs识别大量PAMP的分子基础是我的实验室的主要兴趣所在。最近，我们与David Davies博士(LMB，NIDDK)合作，成功地表达、结晶和确定了TLR3胞外区(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Beta片。这些分子是高度糖基化的，除了马蹄铁的一个侧面完全没有多糖。虽然我们还没有获得TLR3-配体(DsRNA)复合体的高分辨结构，但我们已经通过突变分析定位了dsRNA结合部位。配体结合在TLR3分子无糖的侧面，与组氨酸和天冬酰胺残基结合，靠近C-末端。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通过定义的配体结合位点与dsRNA特异性结合，并且亲和力随着缓冲液酸化和配体大小的增加而增加。TLR3-ECD在溶液中为单体，但与dsRNA结合时形成二聚体。这些二聚体通过成对的两个TLR3-ECD之间的协同作用来稳定，多个TLR3ecd二聚体与长dsRNA结合。与TLR3ecd(40-50bp)形成稳定络合物的最小寡核苷酸每个与一个TLR3ecd二聚体结合，这些也是在细胞中激活TLR3的最小dsRNA。因此，我们已经证明了TLR3信号单位是TLR3分子的配体结合二聚体。展望TLR3之后，我们计划表达和研究来自其他TLR类似物的ECD，以了解它们与TLR3在结构和配体结合功能上的差异。2.核酸PAMPs，如dsRNA、ssRNA和CpG DNA，即TLRs 3、7、8和9的配体，通常隔离在微生物中，只有在病原体内吞和裂解细胞内后才能与TLRs相互作用。相反，TLR 1、2、4、5、6和10与通常存在于介质中的PAMP相互作用，正如预期的那样，这些TLR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在刺激前定位于内质网，并与早期内体中的CpG DNA相互作用。胞内和胞外区域都含有内化信号，我们还在TLR9的胞质区域内定位了两个控制细胞内定位的区域。我们现在正在启动对TLR3细胞内定位的研究。在TLR3的情况下，我们已经建立了激活信号根据细胞类型的不同而不同，我们正在检验信号受到细胞内定位的影响的假设，特别是含有TLR3的细胞内小泡的pH。3.TLRs在获得性免疫中起关键作用，它通过触发DC成熟为具有活性的APC，能够启动幼稚的T细胞。根据我们的观察，DC也表达组胺受体，我们假设组胺对成熟过程有影响。在验证这一假设时，我们发现组胺在TLR诱导的成熟过程中深刻地改变了DC释放的细胞因子，结果是，组胺暴露导致DC将幼稚的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。通过过继转移转基因T细胞的小鼠模型，我们在体内证明了肥大细胞脱颗粒对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，我们现在正在询问这是否对产生的抗体类型有影响

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