Innate Immunity

脊椎动物对感染的免疫反应始于先天免疫系统对病原体的保守分子特征（称为PAMP（病原体相关分子模式））的识别，从而引发立即且通常大规模的炎症反应。先天性反应控制病原体，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PAMP的识别由许多受体介导，其中Toll样受体（TLR）发挥突出作用。与获得性免疫的抗原受体不同，TLR由有限数量的生殖系基因编码，人类中有10个;然而，尽管它们的数量很少，TLR识别非常广泛的PAMP，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识别多种PAMP的分子基础是我实验室的主要兴趣。与大卫戴维斯博士（LMB，NIDDK）合作，我们最近成功地表达，结晶，并确定TLR 3细胞外结构域（ECD）的分子结构。该结构由一个23匝的螺线管组成，弯曲成马蹄形，凹面上有一个大的β片。该分子是高度糖基化的，除了马蹄形的一个侧面完全没有聚糖。虽然我们还没有获得TLR 3-配体（dsRNA）复合物的高分辨率结构，但我们已经通过突变分析定位了dsRNA结合位点。配体在TLR 3分子的无聚糖侧面上结合组氨酸和天冬酰胺残基，靠近C末端。我们现在已经完成了dsRNA寡核苷酸与TLR 3-ECD蛋白相互作用的研究。我们已经发现，纯化的TLR 3-ECD通过确定的配体结合位点特异性结合dsRNA，并且亲和力随着缓冲液酸化和配体大小而增加。TLR 3-ECD在溶液中是单体，但当与dsRNA结合时形成二聚体。这些二聚体通过成对的两个TLR 3-ECD之间的协同相互作用而稳定，并且多个TLR 3ecd二聚体与长dsRNA结合。与TLR 3ecd（40-50 bp）形成稳定复合物的最小寡核苷酸各自结合一个TLR 3ecd二聚体，并且这些也是在细胞中激活TLR 3的最小dsRNA。因此，我们已经证明TLR 3信号单元是TLR 3分子的配体结合的二聚体。展望过去的TLR 3，我们计划表达和检查来自其他TLR旁系同源物的ECD，看看它们在结构和配体结合功能上与TLR 3有何不同。2.核酸PAMP如dsRNA、ssRNA和CpG DNA，TLR 3、7、8和9的配体，通常被隔离在微生物内，并且仅在病原体被胞吞和胞内裂解后才可与TLR相互作用。相比之下，TLR 1、2、4、5、6和10与通常存在于培养基中的PAMP相互作用，并且这些TLR如预期的那样位于细胞表面。因此，正确的细胞定位对于TLR功能至关重要。我们已经研究了TLR 9的定位，以及介导这种定位的TLR 9分子内的基序。我们已经发现，TLR 9位于，在刺激之前，在ER中，并且它与CpG DNA在早期内体中相互作用。细胞质和细胞外结构域都含有内化信号，并且我们还在TLR 9的细胞质结构域内定位了控制细胞内定位的两个区域。我们现在开始研究TLR 3的细胞内定位。在TLR 3的情况下，我们已经确定激活信号根据细胞类型而变化，并且我们正在测试信号传导受细胞内定位影响的假设，特别是含有TLR 3的细胞内囊泡的pH。3. TLR通过触发DC成熟为能够引发初始T细胞的有能力的APC在获得性免疫中起关键作用。基于我们观察到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，我们发现组胺深刻地改变了TLR诱导成熟过程中DC释放的细胞因子，因此，组胺暴露导致DC向Th 2表型转化为幼稚T细胞。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作用于邻近的DC，从而改变免疫应答的性质。通过使用过继转移转基因T细胞的小鼠模型，我们已经证明了肥大细胞脱粒对体内T细胞极化的影响。通过使用缺乏肥大细胞的小鼠，我们现在已经表明肥大细胞影响小鼠中的Th 1/Th 2平衡，并且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