Studies of Chemokine-Receptor Interactions with Chemokines and alarmins

总的来说，我的实验室研究了趋化因子模拟物与g蛋白偶联趋化受体和激活受体的相互作用，从而对炎症、免疫、自身免疫、癌症和痛觉产生影响。我们已经证明，各种抗菌肽（amp）和核结合蛋白模拟趋化因子也有能力快速激活宿主免疫反应。我们建议把这些早期预警信号称为警报。警报器的特点是在体内具有体外趋化作用或对表达GiPCR的细胞具有募集活性，同时具有与其他受体相互作用的能力，导致未成熟的树突状细胞（iDC）被激活，成熟为抗原呈递，T淋巴细胞激活树突状细胞（mDC），从而产生体内免疫佐剂作用。这些警报如果与抗原一起使用，会导致体内细胞和体液对抗原的免疫反应显著增强。我们之前发现α和β两种类型的防御素都是对未成熟树突状细胞（iDCs）具有趋化和激活作用的警报器，并具有体内免疫佐剂作用。一些β防御素与CCR6趋化因子受体相互作用，另一些与CCR2相互作用，而α防御素与一种未知的g蛋白偶联受体（GiPCR）相互作用。另一种抗菌肽被称为cathelicidin （LL37）及其小鼠同源物CRAMP，对单核细胞和iDC前体上表达的FPR2受体具有趋化作用，诱导iDC成熟，并且在体内与明矾一样是有效的佐剂。此外，我们之前还发现嗜酸性粒细胞衍生的神经毒素（EDN，一种核糖核酸酶）、淋巴细胞的颗粒蛋白、中性粒细胞的乳铁蛋白和HMGB1（一种核结合蛋白）都是功能性警报器。虽然警报在结构上是不同的，但它们是从白细胞和上皮细胞颗粒或受损细胞中预先形成并迅速释放的。在促炎刺激物的作用下，胃肠道、胃肠道和气管支气管内的角质形成细胞或上皮细胞也可诱发警报器。因此，警报器可能代表一个早期预警系统，提醒宿主防御危险信号。在过去几年中，我们还发现了一种防御素激活DC的方法。我们发现，人β -防御素2和3通过与自身或细菌来源的DNA（如CpG）结合，以Toll受体样9 （TLR9）依赖的方式成为浆细胞样DC产生干扰素- α (1FNK)的有效兴奋剂，ifn - α反过来在体外和体内诱导其他促炎细胞因子。小鼠皮下注射防御素- dna复合物可显著增强局部炎症反应。腹腔内感染复合物增强了细胞和免疫对同时感染抗原的反应。这些发现使我们提出存在于炎症部位的防御素有助于增强免疫反应，并且它们可以大大增强CpG的疫苗佐剂作用。在过去的四年中，我们还发现并鉴定了高迁移率核小体结合蛋白-1 （HMGN-1）作为细胞外警报蛋白，是脂多糖（LPS）诱导（tlr -4依赖）免疫反应的必要介质。HMGN-1具有在注射部位招募和诱导树突状细胞（DC）成熟的能力。HMGN-1以TLR4依赖的方式激活NF κ B和多种MAP激酶。HMGN-1与抗原共给药后，可显著增强特异性免疫反应，并具有有利于Th1免疫反应的强佐剂作用。相反，通过基因工程导致HMGN-1缺失的小鼠，即使对抗原与LPS一起施用，抗原特异性免疫反应也大大降低。HMGN-1敲除小鼠的这种免疫缺陷与DC在免疫位点募集不足和DC产生的细胞因子减少有关。因此，主要来源于非白细胞（如上皮细胞）的HMGN-1在先天和适应性免疫反应的发展中起着非冗余的关键作用。因此，HMGN-1基因敲除小鼠也显示出比正常小鼠更频繁地自发产生肿瘤，并表现出对肿瘤攻击的抵抗力降低。因此，我们计划利用HMGN-1作为抗肿瘤疫苗佐剂。

Overall, my laboratory investigates the interactions of chemokine mimics with G-protein coupled chemotactic receptors and activating receptors with resultant effects on inflammation, immunity, autoimmunity, cancer and algesia. We have shown that a variety of antimicrobial peptides (AMPs) and nuclear binding proteins that mimic chemokines also have the capacity to rapidly activate host immune responses. We have proposed calling these early warning signals alarmins. Alarmins are characterized by having in vitro chemotactic in vivo or recruitment activity for cells expressing GiPCR, together with the capacity to interact with other receptors resulting in the activation of immature dendritic cells (iDC) to mature into antigen-presenting, T lymphocyte activating dendritic cells (mDC) with resultant in vivo immunoadjuvant effects. These alarmins, if administered together with an antigen, result in considerable augmentation of both in vivo cellular and humoral immune responses to the antigen. We previously identified both alpha and beta types of defensins as alarmins with chemotactic and activating effects on immature dendritic cells (iDCs) and in vivo immunoadjuvant effects. Some of the beta defensins interact with the CCR6 chemokine receptor, others with CCR2, while alpha defensins interact with an as yet unknown G-Protein Coupled Receptors (GiPCR). Another antimicrobial peptide known as cathelicidin (LL37) and its murine homologue CRAMP are chemotactic for FPR2 receptors expressed on monocytes and precursors of iDC, induce the maturation of iDC and are equally as potent adjuvants in vivo as alum. In addition, we have previously also identified eosinophil derived neurotoxin (EDN, a ribonuclease), granulysin from lymphocytes, lactoferrin from neutrophils and HMGB1, a nuclear binding protein, as functional alarmins. Although alarmins are structurally distinct, they are preformed and rapidly released from granules of leukocytes and epithelial cells or from injured cells. Alarmins can also be induced in response to proinflammatory stimulants by keratinocytes or epithelial cells lining the GI tract, GU tract and tracheobronchial tree. As such, alarmins probably represent an early warning system to alert the host defense to danger signals. During the past several years, we have also identified a means by which defensins activate DC. We have found that human beta-defensins 2 and 3, by binding to self or bacterial derived DNA such as CpG, become potent stimulants of plasmacytoid DC production of Interferon-alpha (1FNK) in a Toll Receptor-like 9 (TLR9) dependent manner the IFN-alpha in turn induces other proinflammatory cytokines in vitro and in vivo. Subcutaneus injections in mice of defensin-DNA complexes results in markedly enhanced local inflammatory reactions. Intraperitoneal infections of the complexes augments both cellular and immune responses to concommitantly infected antigens. These findings lead us to propose that defensins present at inflammatory sites serve to amplify immune responses and that they can considerably enhance the vaccine adjuvant effects of CpG. During the past four years, we have also identified and characterized High Mobility Group Nucleosome-binding protein-1 (HMGN-1) as an extracellular alarmin that is a necessary mediator of lipopolysaccharide (LPS)-induced (TLR-4-dependent) immune responses. HMGN-1 has the capacity to recruit and induce the maturation of dendritic cells (DC) at sites of injection. HMGN-1 activates NF kappa B and multiple MAP kinases largely in a TLR4 dependent manner. Upon coadministration with antigens, HMGN-1 markedly enhances specific immune responses and has potent adjuvant effects favoring Th1 immune responses. Conversely, mice genetically engineered to be deficient in HMGN-1 had greatly reduced antigen specific immune responses even in response to antigens administered together with LPS. This immune deficiency of HMGN-1 knockout mice was associated with deficient recruitment of DC to sites of immunization and reduced cytokine production by DC. Thus, HMGN-1 which is largely derived from non-leukocytes (e.g. epithelial cells) plays a non-redundant critical role in the development of innate and adaptive immune responses. Consequently, HMGN-1 knockout mice also have been shown to spontaneously develop tumors more frequently than normal and to exhibit reduced resistance to tumor challenge. We therefore plan to utilize HMGN-1 as an antitumor vaccine adjuvant.