Molecular Analysis Of Leukocyte Activation By Chemoattractants

该项目的目的是确定血液白细胞迁移到发炎或感染的特定组织部位的分子机制和生物学背景。我们已经集中在介导这一过程的趋化蛋白，并确定了一个大家族的趋化受体，部署在白细胞表面的成员。我们还鉴定了由病毒（包括疱疹病毒、痘病毒和HIV）产生的不同化学引诱物和化学引诱物受体模拟物。我们使用基因组学、分子生物学、细胞生物学和流行病学作为分析这些分子的主要方法。一个主要的目标是确定具体的疾病协会个别化学引诱物和化学引诱物受体，以确定潜在的新的治疗靶点。 一个关键的策略是分析疾病模型中基因敲除小鼠的表型以及人类疾病队列中相应人类基因中功能缺失突变的关联。 在2011财年，我们报告了以下领域的发现：1.西尼罗病毒致病机理; 2.动脉粥样硬化; 3.免疫缺陷和4.行为; 5.念珠菌病; 6.鉴定化学引诱物受体基因;和7. MRSA感染。 1.）我们发现的证据表明，单核细胞趋化因子受体Ccr 2是至关重要的生存和选择性炎症单核细胞招募到大脑中的小鼠模型的西尼罗河病毒脑炎。 WNV在完全依赖Ccr 2的模型中诱导选择性单核细胞增多。 我们发现，单核细胞运输到大脑似乎涉及两个Ccr 2依赖的过程：单核细胞从骨髓运输到血液和从血液到大脑。这扩展了我们以前在这一领域的工作，确定CCR 5和OAS 1作为症状性西尼罗河病毒感染的保护因素。最近的工作是重要的，因为它提供了新的见解CCR 2补充CCR 5控制症状性WNV疾病的机制。新的数据表明，如果接受治疗的患者感染了西尼罗河病毒，用药物阻断CCR 2可能会增加症状性西尼罗河病毒疾病的风险。 2.）我们在基于饮食和ApoE缺乏的小鼠模型中发现了Ccr 6正调控动脉粥样硬化形成的证据。 在该模型中，与其他已建立的趋化因子受体相比，Ccr 6的贡献很强。 其机制可能与Ccr 6对ApoE基因缺陷小鼠单核细胞增多的调控和Ccr 6对单核细胞趋化性的调节有关。 我们发现，原代小鼠单核细胞表达功能性Ccr 6。 骨髓移植研究证实，Ccr 6在模型中对造血细胞起作用，但这项工作留下了Ccr 6可能在模型中以单核细胞以外的其他类型白细胞水平发挥作用的可能性。 这一发现的重要性在于将Ccr 6确定为动脉粥样硬化性心血管疾病药物开发的潜在靶点。 3.）第三章我们发现了两个兄弟姐妹的孩子与一个新发现的孟德尔疾病称为G6 PC 3缺乏症。 我们发现，骨髓病变是这种疾病的一个特征，这可能部分解释了严重的中性粒细胞减少症和对细菌病原体感染的易感性增加。 我们发现，髓样病变可能是由于中性粒细胞上CXCR 4（一种已知的中性粒细胞骨髓归巢受体）表达增加所致。 我们还报道了FDA批准的药物Mozobil（AMD 3100）在G6 pc 3缺乏症小鼠中作为中性粒细胞动员剂是完全有效的，并且它在大多数WHIM综合征患者中发现的CXCR 4功能突变形式中也是完全活跃的，WHIM综合征也是一种以骨髓增生异常为特征的疾病。我们有一项已批准的开放I期方案，用于研究释倍灵在WHIM综合征患者中的安全性和疗效。 4.）我们发现缺乏趋化因子受体Fpr 1的小鼠是过度活跃的。 小鼠似乎具有受损的恐惧记忆和焦虑样行为，这可能是由于HPA轴的异常调节导致皮质酮水平增加，皮质酮是小鼠中焦虑样行为的已知修饰剂。这是第一个描述白细胞化学引诱物受体在行为中的功能作用的工作，并表明这些受体在免疫系统之外具有多任务功能。 5.）我们报道了免疫系统对系统性念珠菌攻击的反应是高度器官特异性的，肾脏单独不能控制感染和免疫病理学。 接受相等念珠菌负荷的其他器官被中性粒细胞浸润，但以短暂的、非病理性的和高度特异质的方式。 6.）我们的特点是一个新的和非典型的FPR家族成员的趋化受体基因。 命名为Fpr-rs 8的基因是Fpr-rs 2的重复，但明显截短。 我们描述了这两个基因的不同进化历史，并认为Fpr-rs 8不是假基因，因为它是转录和调节的，蛋白质似乎是制造的，缺乏它的小鼠寿命缩短了30%。 fpr-rs 8似乎局限于细胞内空间，可能不发出信号。 7.） 我们还发现了病原体利用宿主防御因子诱导自身毒力因子的机制。 特别是S.金黄色葡萄球菌能够结合诱导毒力因子葡萄球菌蛋白A释放的特异性宿主趋化因子。 这是在体外定义的，但我们有数据证明其在S小鼠模型中的生物相关性。金黄色脓肿形成和患者样本。 8.） 最后，我们发表了一份初步报告，确定自我管理的过度通气是一种快速，免费，易于实施，安全和高效的治疗方法，适用于患有严重运动相关肌肉痉挛的患者，将他们从睡眠中唤醒。 该技术也适用于其他形式的抽筋。

The aim of this project is to define the molecular mechanisms and biological contexts for blood leukocyte migration to specific tissue sites that are inflamed or infected. We have focused on chemoattractant proteins that mediate this process and have identified members of a large family of chemoattractant receptors that are deployed on the leukocyte cell surface. We have also identified members of a diverse group of chemoattractant and chemoattractant receptor mimics made by viruses, including herpesviruses, poxviruses and HIV. We use genomics, molecular biology, cell biology and epidemiology as the principle methods for analyzing these molecules. A major goal is to identify specific disease associations of individual chemoattractant and chemoattractant receptors, in order to identify potential new therapeutic targets. A key strategy is to analyze phenotypes of gene knockout mice in disease models as well as associations of loss of function mutations in the corresponding human genes in human disease cohorts. In FY11 we reported discoveries in the following areas: 1. West Nile virus pathogenesis; 2. atherosclerosis; 3. immunodeficiency and 4. behavior; 5. candidiasis; 6. identifying chemoattractant receptor genes; and 7. MRSA infection. 1.) We found evidence that the monocyte chemokine receptor Ccr2 is critical for survival and for selective inflammatory monocyte recruitment to brain in a mouse model of WNV encephalitis. WNV induces a selective monocytosis in the model that is completely Ccr2 dependent. We found that monocyte trafficking into the brain appears to involve two Ccr2-dependent processes: monocyte trafficking from bone marrow to blood and from blood to brain. This extends our previous work in this area that identified CCR5 and OAS1 as protective factors for symptomatic WNV infection. The recent work is important because it provides new insight into the mechanism by which CCR2 complements CCR5 in control of symptomatic WNV disease. The new data suggest that blocking CCR2 with a drug may increase the risk of symptomatic WNV disease should treated patients become infected with WNV. 2.) We found evidence that Ccr6 positively regulates atherogenesis in a mouse model based on diet and ApoE deficiency. The contribution of Ccr6 was strong compared to other established chemokine receptors in this model. The mechanism may involve Ccr6 control of monocytosis in ApoE deficient mice and Ccr6 regulation of monocyte chemotaxis. We showed that primary mouse monocytes express functional Ccr6. Bone marrow transfer studies established that Ccr6 is operating on a hematopoietic cell in the model, but the work left open the possibility that Ccr6 may function in the model at the level of other types of leukocytes besides monocytes. The importance of the finding is in identifying Ccr6 as a potential target for drug development in atherosclerotic cardiovascular disease. 3.) We identified two sibling children with a newly discovered Mendelian disease called G6PC3 deficiency. We showed that myelokathexis is a feature of this disease, which may explain in part the severe neutropenia and increased susceptibility to infection by bacterial pathogens. We found that the myelokathexis may result from increased expression on neutrophils of CXCR4 a known bone marrow home receptor for neutrophils. We also reported that the FDA-approved drug Mozobil (AMD3100) is fully effective as neutrophil mobilizing agent in mice with G6pc3 deficiency, and that it is also fully active at the mutant gain of function form of CXCR4 found in most patients with WHIM syndrome, a disease also characterized by myelokathexis. We have an approved open Phase 1 protocol to study the safety and efficacy of Mozobil in patients with WHIM syndrome. 4.) We found that mice lacking the chemoattractant receptor Fpr1 are hyperactive. The mice appear to have impaired fear memory and anxiety like behavior, and this may be due to abnormal regulation of the HPA axis resulting in increased corticosterone levels, a known modifier of anxiety like behavior in mice. This is the first work describing a functional role of a leukocyte chemoattractant receptor in behavior, and suggests that these receptors multitask outside of the immune system. 5.) We reported that the response of the immune system to systemic candida challenge is highly organ specific, with kidney alone unable to control the infection and the immunopathology. Other organs receiving an equal candida burden become infiltrated by neutrophils but in a transient, non-pathologic and highly idiosyncratic manner. 6.) We characterized a novel and atypical member of the FPR family of chemoattractant receptor genes. The gene named Fpr-rs8 is a duplication of Fpr-rs2, but is markedly truncated. We describe distinct evolutionary histories of the two genes, and argue that Fpr-rs8 is not a pseudogene, because it is transcribed and regulated, the protein appears to be made, and mice lacking it have a 30% shortened life-span. Fpr-rs8 appears to be restricted to the intracellular space and probably does not signal. 7.) We also discovered a mechanism by which a pathogen exploits a host defense factor to induce its own virulence factor. In particular, S. aureus is able to bind specific host chemokines which induce release of the virulence factor Staph protein A. This was defined in vitro but we have data attesting to its biological relevance in mouse models of S. aureus abscess formation and patient samples. 8.) Lastly, we published a preliminary report identifying self-administered hyperventilation as a quick-acting, free, easy to implement, safe and highly effective treatment for patients suffering from severe exercise-associated muscle cramping awakening them from sleep. The technique may also work for other forms of cramping.