Studies of Receptor Interactions and Effects of Alarmins

受体相互作用和警报素作用的研究

• 批准号: 8937677
• 负责人: JOOST J OPPENHEIM
• 金额: $ 99.18万
• 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/8937677
• 关键词: Adjuvant; Anthrax Vaccines; Anthrax disease; Antibodies; Antigen Presentation Pathway; Antigen-Presenting Cells; Antigens; Bacillus (bacterium); Binding Proteins; CCR6 gene; Cancer Vaccines; Cell Line; Cell Maturation; Cell Nucleus; Cells; Colonic Neoplasms; Cytoplasmic Granules; Cytosol; Defensins; Dendritic Cells; Development; Eosinophil-Derived Neurotoxin; Epithelial Cells; Exhibits; G-Protein-Coupled Receptors; Gastrointestinal tract structure; Genetically Engineered Mouse; Growth; HMGB1 gene; HMGN1 Protein; HMGN1 gene; Homologous Gene; Host Defense; Immune; Immune response; Immunization; Immunoglobulin G; Immunologic Adjuvants; Immunosuppressive Agents; In Vitro; Infection; Inflammation; Injection of therapeutic agent; Injury; Knockout Mice; Lactoferrin; Leukocytes; Link; Lipopolysaccharides; Lymphocyte; Mediator of activation protein; Mitogen-Activated Protein Kinases; Mus; NF-kappa B; Nuclear; Organism; Play; Polyglutamic Acid; Production; Property; Proteins; Recombinants; Recruitment Activity; Resistance; Ribonucleases; Role; Signal Transduction; Site; System; T-Cell Activation; T-Lymphocyte; TLR4 gene; Technology; Tissues; Tracheobronchial; Trees; Tumor Antigens; Tumor Immunity; Vaccines; Virulence Factors; alpha-Defensins; aluminum sulfate; anthrax toxin; antimicrobial peptide; base; beta-Defensins; capsule; cathelicidin; chemokine; chemokine receptor; cytokine; extracellular; gene gun; granulysin; immunogenic; improved; in vivo; inhibitor/antagonist; injured; intraperitoneal; keratinocyte; melanoma; microbial; monocyte; neoplastic cell; neutrophil; overexpression; plasmid DNA; receptor; research study; response; therapeutic vaccine; tumor; tumor growth

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 warnings signals alarmins. Alarmins are characterized by having in vitro chemotactic or in vivo recruitment activity for cells expressing GiPCR, together with the capacity to interact with other receptors resulting in the activation of immature dendritic cells (iDC) into mature antigen-presenting capable of interacting with T lymphocytes. 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. Cathelicidins also induce the maturation 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 constitutively available. Alarmin are rapidly released from granules, cytosol or nucleus of leukocytes and epithelial cells or from injured cells. Alarmins can also be induced to be produced 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 four years, we have also identified and characterized High Mobility Group Nuclesome-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. In a previous experimental study, we showed that intraperitoneal administration of anthrax vaccine with HMGN1 protein markedly increased the production of both primary and secondary protective IgG antibodies to anthrax toxin. During this past year we evaluated the properties of the antiphagocytic capsule consisting of polyglutamic acid, of Bacillus anthraces. This capsule is a major virulence factor and is an immunosuppressive inhibitor of dendritic cell maturation. We plan to couple HMGN-1 to this capsular antigen, because a mixture of the two proteins becomes an immunogenic stimulant and may potentially provide a better vaccine against anthrax. During the past year we also showed that HMGN-1 knockout mice exhibit reduced resistance to tumor (EG-7 or EL-4) challenge. Conversely,tumor cells (EG-7 or EL-4) when transfected to overexpress HMGN1 showed a marked reduction in the rate of growth in normal mice. These observations indicated that HMGN1 was capable of augmenting tumor immunity. Since HMGN1 is the most potent of the alarmins in inducing antigen specific immune responses, we have chosen to study it further as an antitumor adjuvant. To maximize the adjuvant effects of HMGN1, we covalently linked it to a gp100 melanoma tumor antigen. Antigens fused to adjuvants have been shown to be delivered more effectively to the appropriate intracellular compartments of antigen presenting cells (APC's) resulting in improved antigen processing and presentation and greater T cell activation. We have immunized mice with gp100 linked to HMGN1 in the form of plasmid DNA using gene gun technology. This succeeded in inducing about 70% of the immunized mice to be resistant to a challenge with B16 melanoma tumor cells. However, therapy of mice with this plasmid DNA, which had been injected with B16 melanoma tumor cells four days previously, failed to inhibit tumor growth. We therefore injected a recombinant HMGN1 protein directly intratumorally into CT26 colon tumors in mice to proximate the adjuvant and antigen. This therapeutic vaccine trial did have a significant beneficial effect in slowing the tumor growth and prolonging the survival of mice, but did not cure any of the mice. We have to improve the delivery of the tumor vaccine and employ it in conjunction with other antitumor therapies.

我们已经表明，各种抗菌肽（AMP）和核结合蛋白，模拟趋化因子也有能力迅速激活宿主的免疫反应。我们建议称这些早期预警信号为危言耸听。Alarmins的特征是对表达GiPCR的细胞具有体外趋化或体内募集活性，以及与其他受体相互作用的能力，从而将未成熟的树突状细胞（iDC）激活为能够与T淋巴细胞相互作用的成熟抗原提呈细胞。这些警报素如果与抗原一起施用，则导致对抗原的体内细胞和体液免疫应答的显著增强。我们先前鉴定了α和β型防御素作为对未成熟树突状细胞（iDC）具有趋化和活化作用以及体内免疫佐剂作用的警报素。一些β防御素与CCR 6趋化因子受体相互作用，另一些与CCR 2相互作用，而α防御素与迄今未知的G蛋白偶联受体（GiPCR）相互作用。另一种被称为凯萨林菌素（LL 37）的抗微生物肽及其鼠同源物CRAMP对单核细胞和iDC前体上表达的FPR 2受体具有趋化性。Cathelicidins还诱导成熟iDC，并且在体内与明矾一样是有效的佐剂。此外，我们以前也确定了嗜酸性粒细胞衍生的神经毒素（EDN，核糖核酸酶），颗粒溶素从淋巴细胞，乳铁蛋白从中性粒细胞和HMGB 1，核结合蛋白，作为功能性报警。虽然alarmin在结构上是不同的，但它们是预先形成的，并且组成型可用。Alarmin从白细胞和上皮细胞的颗粒、胞浆或细胞核或从损伤细胞中迅速释放。Alarmins也可以被诱导以响应于由衬于GI道、GU道和气管支气管树的角质形成细胞或上皮细胞产生的促炎刺激物。因此，警报器可能代表一种早期预警系统，以提醒宿主防御危险信号。在过去的四年中，我们还确定和表征了高迁移率族核小体结合蛋白-1（HMGN-1）作为一种细胞外警报素，它是脂多糖（LPS）诱导的（TLR-4依赖性）免疫应答的必要介质。HMGN-1具有在注射部位募集和诱导树突状细胞（DC）成熟的能力。HMGN-1主要以TLR 4依赖性方式激活NF κ B和多种MAP激酶。与抗原共同给药后，HMGN-1显著增强特异性免疫应答，并具有有利于Th 1免疫应答的有效佐剂作用。相反，基因工程改造为HMGN-1缺陷的小鼠即使在对与LPS一起施用的抗原的应答中也具有大大降低的抗原特异性免疫应答。HMGN-1基因敲除小鼠的这种免疫缺陷与DC向免疫位点的募集不足和DC产生的细胞因子减少有关。因此，主要来源于非白细胞（例如上皮细胞）的HMGN-1在先天性和适应性免疫应答的发展中起着非冗余的关键作用。在以前的实验研究中，我们表明，腹腔注射炭疽疫苗与HMGN 1蛋白显着增加生产的初级和二级保护性IgG抗体炭疽毒素。在过去的一年中，我们评估了由聚谷氨酸组成的抗吞噬胶囊的特性，炭疽芽孢杆菌。这种荚膜是一种主要的毒力因子，是树突状细胞成熟的免疫抑制剂。我们计划将HMGN-1与这种荚膜抗原偶联，因为这两种蛋白质的混合物成为免疫原性刺激物，并可能提供更好的炭疽疫苗。在过去的一年中，我们还发现HMGN-1基因敲除小鼠对肿瘤（EG-7或EL-4）攻击的抵抗力降低。相反，肿瘤细胞（EG-7或EL-4）转染过表达HMGN 1时，在正常小鼠的生长速度显着下降。这些观察结果表明，HMGN 1能够增强肿瘤免疫。由于HMGN 1是在诱导抗原特异性免疫应答中最有效的警报素，我们选择进一步研究其作为抗肿瘤佐剂。为了使HMGN 1的佐剂效应最大化，我们将其与gp 100黑色素瘤肿瘤抗原共价连接。已显示与佐剂融合的抗原更有效地递送至抗原呈递细胞（APC）的适当细胞内区室，导致改进的抗原加工和呈递以及更大的T细胞活化。我们利用基因枪技术，以质粒DNA的形式将与HMGN 1连接的gp 100免疫小鼠。这成功地诱导了约70%的免疫小鼠对B16黑色素瘤肿瘤细胞的攻击具有抗性。然而，用这种质粒DNA治疗四天前注射了B16黑色素瘤肿瘤细胞的小鼠，未能抑制肿瘤生长。因此，我们将重组HMGN 1蛋白直接瘤内注射到小鼠的CT 26结肠肿瘤中以接近佐剂和抗原。这种治疗性疫苗试验确实在减缓肿瘤生长和延长小鼠生存期方面具有显著的有益效果，但没有治愈任何小鼠。我们必须改进肿瘤疫苗的递送，并将其与其他抗肿瘤疗法结合使用。