Molecular Analysis Of Leukocyte Activation By Chemoattractants

这个项目的目的是确定血液白细胞迁移到炎症或感染的特定组织部位的分子机制和生物学背景。我们一直专注于介导这一过程的趋化蛋白，并鉴定了部署在白细胞表面的趋化受体大家族的成员。我们还鉴定了一组不同的趋化物质和趋化物质受体模拟物，包括疱疹病毒、痘病毒和艾滋病毒。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单个趋化物质和趋化物质受体的特定疾病关联，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功能突变丧失的相关性。在2011财年，我们报告了以下方面的发现：1.西尼罗河病毒的发病机制；2.动脉粥样硬化；3.免疫缺陷和4.行为；5.念珠菌病；6.确定趋化受体基因；7.MRSA感染。 1)我们发现有证据表明，单核细胞趋化因子受体CCR2对于WNV脑炎小鼠模型的生存和选择性炎性单核细胞募集到脑内至关重要。WNV在完全依赖CCR2的模型中诱导选择性单核细胞增多。我们发现，单核细胞转运到大脑似乎涉及两个依赖CCR2的过程：单核细胞从骨髓转运到血液和从血液转运到脑。这是我们之前在这一领域的工作的扩展，该工作将CCR5和OAS1确定为有症状的西尼罗河病毒感染的保护因素。最近的工作很重要，因为它为CCR2补充CCR5控制症状性西尼罗河病毒疾病的机制提供了新的见解。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CCR2可能会增加出现症状的西尼罗河病毒病的风险。 2.)我们发现有证据表明，Ccr6对基于饮食和载脂蛋白E缺乏的小鼠模型的动脉粥样硬化有积极的调节作用。在这个模型中，与其他已建立的趋化因子受体相比，Ccr6的贡献很大。其机制可能涉及Ccr6对载脂蛋白E缺陷小鼠单核细胞增多的调控和Ccr6对单核细胞趋化的调节。我们发现原代小鼠单核细胞表达有功能的Ccr6。骨髓移植研究证实，Ccr6在模型中的造血细胞上发挥作用，但这项工作留下了一种可能性，即Ccr6可能在模型中发挥单核细胞以外的其他类型白细胞的功能。这一发现的重要性在于确定Ccr6是动脉粥样硬化性心血管疾病药物开发的潜在靶点。 3.)我们确定了两个兄弟姐妹患有一种新发现的孟德尔疾病，称为G6PC3缺乏症。我们发现骨髓异常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和对细菌病原体感染的易感性增加。我们发现，骨髓病变可能是由于中性粒细胞上CXCR4的表达增加所致，CXCR4是已知的中性粒细胞的骨髓宿主受体。我们还报道了FDA批准的药物MOZOBIL(AMD3100)在G6pc3缺乏的小鼠中作为中性粒细胞动员剂完全有效，并且在大多数突发综合征患者中发现的CXCR4功能形式的突变获得也是完全有效的，这种疾病的特征也是骨髓病。我们已经批准了一项开放的第一阶段方案来研究MOZOBIL在突发事件综合征患者中的安全性和有效性。 4.)我们发现，缺乏化学吸引受体FPR1的小鼠是过度活跃的。小鼠的恐惧记忆和类焦虑行为似乎受到了损害，这可能是由于HPA轴的异常调节导致皮质酮水平上升，皮质酮是小鼠焦虑类行为的已知修饰物。这是第一项描述白细胞趋化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项任务。 5.)我们报道，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，仅靠肾脏无法控制感染和免疫病理。其他接受相同念珠菌负担的器官会被中性粒细胞渗透，但以一种短暂的、非病理的和高度特异的方式。 6.)我们鉴定了一个新的和非典型的趋化受体基因家族的成员。名为fpr-rs8的基因是fpr-rs2的复制，但明显被截断。我们描述了这两个基因的不同进化历史，并认为fpr-rs8不是一个假基因，因为它是转录和调控的，蛋白质似乎是合成的，缺乏它的小鼠寿命缩短了30%。Fpr-Rs8似乎仅限于细胞内空间，可能没有信号。 7.)我们还发现了一种机制，病原菌利用宿主防御因子来诱导自己的毒力因子。特别是，金黄色葡萄球菌能够结合特定的宿主趋化因子，诱导毒力因子葡萄球菌蛋白A的释放。这是在体外定义的，但我们有数据证明它在金黄色葡萄球菌脓肿形成的小鼠模型和患者样本中具有生物学相关性。 8.)最后，我们发表了一份初步报告，确定自我管理过度换气是一种快速、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性肌肉痉挛的患者，将他们从睡眠中唤醒。这项技术也可能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.