Studies of Chemokine-Receptor Interactions with Chemokines and alarmins

总体而言，我的实验室研究趋化因子类似物与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、癌症和疼痛产生影响。我们已经证明，各种模拟趋化因子的抗菌肽(AMP)和核结合蛋白也具有快速激活宿主免疫反应的能力。我们提议将这些早期预警信号称为警报。Alarmins的特征是在体内具有体外趋化活性或对表达GiPCR的细胞的募集活性，以及与其他受体相互作用的能力，从而激活未成熟的树突状细胞(IDC)成熟为抗原提呈，T淋巴细胞激活树突状细胞(MDC)，从而产生体内免疫佐剂效应。这些警报如果与抗原一起使用，会导致体内对该抗原的细胞和体液免疫反应显著增强。我们先前发现α和β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以及体内免疫佐剂作用的警戒素。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，而阿尔法防御素与一种未知的G蛋白偶联受体(GiPCR)相互作用。另一种抗菌肽长春花素(LL37)及其小鼠同源基因对表达在单核细胞和IDC前体细胞上的FPR2受体具有趋化作用，可诱导IDC成熟，在体内与明胶一样具有强大的佐剂作用。此外，我们还鉴定了嗜酸性粒细胞源性神经毒素(EDN，一种核糖核酸酶)、淋巴细胞颗粒溶素、中性粒细胞乳铁蛋白和核结合蛋白HMGB1为功能性警报蛋白。虽然Alarmins在结构上是不同的，但它们是预先形成的，并从白细胞颗粒和上皮细胞或损伤细胞中迅速释放出来。此外，胃肠道、胃肠道和气管支气管树上的角质形成细胞或上皮细胞也可以对促炎症刺激作出反应，从而诱导警报器的产生。因此，警报器可能代表了一种早期预警系统，以提醒宿主防御系统注意危险信号。在过去的几年里，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(Toll Receptor-like 9，TLR9)依赖的方式成为血浆细胞样树突状细胞产生干扰素-α(1FNK)的强大刺激剂，而干扰素-α在体外和体内反过来又诱导其他促炎细胞因子。小鼠皮下注射防御素-DNA复合体可显著增强局部炎症反应。腹膜腔内感染该复合体可增强细胞和免疫对感染抗原的反应。这些发现导致我们提出，存在于炎症部位的防御素有助于放大免疫反应，并且它们可以显著增强CpG的疫苗佐剂效果。在过去的四年里，我们还鉴定了高迁移率族核小体结合蛋白-1(HMGN-1)，它是脂多糖(LPS)诱导的(TLR-4依赖的)免疫反应的必要中介。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主要以TLR4依赖的方式激活核因子-kappaB和多个MAP激酶。当HMGN-1与抗原联合给药时，显著增强特异性免疫反应，并具有强大的佐剂效应，有利于Th1免疫反应。相反，通过基因工程获得HMGN-1缺陷的小鼠，即使在与脂多糖一起注射抗原的情况下，也会极大地降低抗原特异性免疫反应。HMGN-1基因敲除小鼠的这种免疫缺陷与DC在免疫部位的募集不足和DC产生的细胞因子减少有关。因此，HMGN-1主要来源于非白细胞(如上皮细胞)，在先天性和获得性免疫反应的发展中起着非冗余的关键作用。因此，HMGN-1基因敲除小鼠也被证明比正常小鼠自发发展肿瘤的频率更高，并且对肿瘤挑战的抵抗力降低。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.