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2013财年，我们报告了以下领域的发现：1.白内障发病机制；2.侵袭性念珠菌病；3.动脉粥样硬化。 1)在2013财年，我们继续我们在2011财年和2012财年发表的关于侵袭性念珠菌病小鼠模型器官特异性免疫的工作，在该模型中，所有研究的器官最初都被感染到类似的水平；然而，只有肾脏不能解决感染，并被不受控制的病原体生长和无效的中性粒细胞聚集的组合破坏，占该模型中死亡的原因。在2011财年，我们发表了一篇关于该模型免疫病理学的描述性论文，在2012财年，我们发表了一篇机械论文，定义了趋化因子受体CCR1在该模型中介导中性粒细胞依赖的免疫病理学和死亡中的重要作用。现在，在2013财年，我们已经确定CX3CR1是一个关键的趋化因子受体，通过促进肾脏巨噬细胞的积累来促进模型中真菌的早期清除和存活。CX3CR1的主要作用机制可能是通过造血细胞和非造血细胞(或非放射抗性细胞)发挥作用，而在表达CX3CR1的模型中，单核细胞和巨噬细胞是肾脏的主要造血细胞。我们发现，在模型中，CX3CR1促进了肾脏巨噬细胞的存活，但似乎对细胞的杀伤能力或它们进入肾脏的运输并不重要。这是首次发现调节单核/巨噬细胞在侵袭性念珠菌病中的作用的关键分子因素。到目前为止，我们在模型中的工作突出了中性粒细胞和巨噬细胞之间的微妙平衡，以控制由不同的趋化因子受体介导的念珠菌，以及如果病原体或免疫细胞没有得到适当的调控，会导致死亡的有害影响。这项最新的工作意义重大，因为它发现人类由于M280等位基因的遗传而导致CX3CR1缺陷，在侵袭性念珠菌病的背景下，不良结局的风险增加。 2.)在2013财年，我们发表了第一篇论文，鉴定了趋化因子FPR1在人晶状体上皮细胞上的表达。之所以开展这项工作，是因为我们在FPR1基因敲除小鼠的衰老过程中发现了严重的白内障(尚未发表)。这种表型与晶状体中的炎症或感染无关，因此我们询问晶状体细胞是否也能表达该受体。晶状体上皮细胞系的获得为这项工作提供了便利，本论文基本上是从基因、RNA、蛋白质和功能水平上对这些细胞上的受体进行生化分析。尽管我们在所有水平上都发现了该受体的证据，但与白细胞中FPR1的特性相比，它的特性是不典型的。特别是，表达水平很低，而且大多数结合位点似乎是非特异性的。然而，FACS的表面表达似乎很高，细胞对FPR1激动剂做出反应的信号转导能力也很高。这一发现的意义在于，它为FPR1在晶状体维护中的直接作用提供了分子上的可能性，这与我们未发表的FPR1 Ko小鼠的白内障表型一致。 3.)在2013财年，我们继续我们关于趋化因子调节动脉粥样硬化的长期项目。我们发现，趋化因子受体CCR7在缺乏载脂蛋白E的动脉粥样硬化小鼠的这一过程中起着关键的控制作用。我们发现CCR7通过表达造血细胞和调节T细胞在血管壁的积聚来发挥作用。这一发现的意义在于，它确定了在血管壁的获得性免疫反应中重要的分子因素，并确定了在疾病中调节的潜在靶点。 在2013财年，我们还为NIH同事发起的合作工作做出了实质性贡献，这些工作涉及Fpr2在结肠炎和结肠炎相关肿瘤发生中的作用，FPR1在控制弓形虫感染中的生态失调和死亡率以及控制肝脏李斯特菌感染中的作用，以及缺乏证据表明CCR5缺乏调节严重脊髓灰质炎感染的风险。 在2013财年，我们还组织和开发了关于趋化因子受体的主要综述，包括开发非典型趋化因子受体的新命名。

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 chemoattractants 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 FY13 we reported discoveries in the following areas: 1. Cataract pathogenesis; 2. invasive candidiasis; 3. atherosclerosis. 1.) In FY13, we continued our work published in FY11 and FY12 on organ specific immunity in a mouse model of invasive candidiasis, in which the all organs studied are infected to similar levels initially; however, only kidney cannot resolve the infection and is destroyed by a combination of uncontrolled pathogen growth and ineffective neutrophil accumulation, accounting for death in the model. In FY11, we published a descriptive paper about the immunopathology of the model and in FY12 a mechanistic paper defining an important role for chemokine receptor Ccr1 in mediating neutrophil-dependent immunopathology and death in the model. Now in FY13, we have identified Cx3cr1 as a critical chemokine receptor that promotes early fungal clearance and survival in the model by promoting accumulation of kidney macrophages. The main mechanism appears to be exerted through both hematopoietic and non-hematopoietic (or non-radio-resistant) cells, and monocytes and macrophages are the main hematopoietic cells in the kidney in the model that express Cx3cr1. We found that Cx3cr1 promotes survival of kidney macrophages in the model but does not appear to be important in killing capacity of the cell or their trafficking into the kidney. This is the first identification of a key molecular factor regulating the role of monocytes/macrophages in invasive candidiasis. Together our work to date in the model highlights the delicate balance between neutrophils and macrophages to control Candida mediated by distinct chemokine receptors, and the harmful effects that occur leading to mortality if either pathogen or immune cells are not regulated properly. The latest work is significant because it identifies humans with defective Cx3cr1 due to inheritance of the M280 allele as having increased risk of poor outcome in the context of invasive candidiasis. 2.) In FY13, we published the first paper identifying expression of chemoattractant Fpr1 on human lens epithelial cells. The work was undertaken because of our discovery of severe cataracts in Fpr1 knockout mice during ageing (not yet published). The phenotype was not related to inflammation or infection in the lens, so we asked whether lens cells could also express the receptor. The availability of lens epithelial cell lines facilitated the work, and the paper is basically a biochemical analysis of the receptor on these cells at the gene, RNA, protein and functional levels. Although we found evidence for the receptor at all levels, its properties were atypical compared to the properties of Fpr1 in leukocytes. In particular, expression is low and most of the binding sites appear to be non-specific. Nevertheless, surface expression by FACS appears high as does the ability of the cell to transduce signals in response to Fpr1 agonists. The significance of this finding is that it provides molecular plausibility for a direct role for Fpr1 in lens maintenance consistent with our unpublished cataract phenotype in Fpr1 ko mice. 3.) In FY13, we continued our long-term project on chemokine regulation of atherosclerosis. We found that the chemokine receptor Ccr7 is critical in control of this process in atherosusceptible mice lacking ApoE fed a high fat diet. We found that Ccr7 operated through expression on hematopoietic cells and regulated accumulation of T cells in the vessel wall. The significance of this finding is that it identifies a molecular factor important in the adaptive immune response in the vessel wall and identifies a potential target for modulation in the disease. In FY13 we also made substantive contributions to collaborative work initiated by NIH colleagues on the role of Fpr2 in colitis and colitis associated tumorigenesis, the role of Fpr1 in control of dysbiosis and mortality in T. gondii infection and in control of Listeria infection in the liver, and the lack of evidence that CCR5 deficiency modulates risk of severe polio infection. In FY13, we also organized and developed a major review on chemokine receptors including development of a new nomenclature for atypical chemokine receptors.