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2012财年，我们报告了以下领域的发现：1.突发综合征；2.侵袭性念珠菌病；3.克氏锥虫感染；4.胃肠道微生物群。 1)我们发表了三篇关于严重联合免疫缺陷障碍心血来潮综合征的临床研究的论文。 A.首次描述了一项关于特定CXCR4拮抗剂plerixafor治疗3名成人突发综合征患者的安全性的第一阶段研究结果，该综合征是由CXCR4功能突变的获得引起的。我们发现，在剂量递增的形式下，该药物在7天内是安全的，直到FDA批准的原始适应症的剂量，即癌症移植患者的干细胞动员。此外，我们发现，该药物迅速动员到血液中，几乎所有患者缺乏的白细胞亚群。动员是暂时的，遵循药物的药代动力学，并且在7天的试验中是剂量依赖的。这些结果证明了在最低有效剂量下测试该药物的慢性安全性和血液学有效性，并最终测试其临床疗效(疣消退，减少感染发生率)。 第二篇论文描述了第一个导致突发奇想综合征的错义突变E343K。所有以前的CXCR4突变都是C-尾部截断突变，包括终止于密码子343的E343X。我们从一个家族中鉴定出三名有这种突变的患者，并证明在CXCR4激动剂CXCL12刺激下，白细胞中的信号增加。这一发现表明，所有由CXCR4突变引起的突发综合征病例都可能是由于E343基因的缺失或突变所致。 C.第三篇论文是与LVD的Alison McBride合作完成的，报告了第10例已知的多瘤病毒，从一名突发症状患者的疣样本中分离出来。这为多瘤病毒与人乳头瘤病毒在反复无常综合征疣发病机制中的作用提出了新的问题。 2.)我们报道了IL-10在小鼠模型中对控制克氏毛滴虫致病至关重要。我们通过比较敏感和耐药小鼠感染这种病原体的临床、微生物学、免疫学和分子相关性，确定了IL-10为靶标。然后我们测试了IL-10 KO小鼠，发现一致的死亡率和未控制的寄生虫病与IL-10的T细胞表达有关。非T细胞来源也被牵连，但没有确定。 3.)我们发现，小鼠的肠道内容物，无论是在大肠还是在小肠，都含有强大而有效的白细胞趋化和激活因子。这些活性似乎是由于大肠和小肠中不同的因素造成的，以防不是一种蛋白质。特别是，N-甲酰肽是由所有细菌产生的，是吞噬细胞上表达的特定受体的强大激活因子，不能解释FPR1 KO小鼠评估的活性。这项工作意义重大，因为肠道含有大量细菌，这些细菌产生的因子可能在健康和疾病中与肠道免疫系统相互作用，特别是在炎症性肠病中。对这些因素的了解可能会导致IBD的新疗法。 4.)我们在侵袭性念珠菌病的小鼠模型中定义了器官特异性免疫，在该模型中，所有研究的器官最初都被感染到相似的水平。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，这在该模型中是死亡的原因。我们发现致命性的中性粒细胞依赖的免疫病理依赖于趋化因子受体CCR1，但这并不影响念珠菌的控制。这种效应似乎是由于肾脏中的CCR1激动剂直接作用于血液中性粒细胞CCR1，该细胞在模型后期表达，同时观察到依赖CCR1的中性粒细胞募集。这些结果突出了控制念珠菌所需的中性粒细胞与不受控制的中性粒细胞聚集和激活对肾脏组织的有害影响之间的微妙平衡。这项工作意义重大，因为它确定了CCR1是影响肾脏的侵袭性念珠菌病的潜在药物靶点。

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 FY12 we reported discoveries in the following areas: 1. WHIM syndrome; 2. invasive candidiasis; 3. Trypanosoma cruzi infection; 4. the gastrointestinal tract microbiome. 1.) We published three papers related to clinical studies of the severe combined immunodeficiency disorder WHIM syndrome. a. The first described results from a Phase 1 study of the safety of plerixafor, a specific CXCR4 antagonist, in the treatment of 3 adult patients with WHIM syndrome, which is caused by gain of function mutations in CXCR4. We found in a dose escalation format that the drug was safe for 7 days up to the dose approved by the FDA for the original indication, stem cell mobilization in patients being transplanted for cancer. In addition, we found that the drug rapidly mobilized into the blood almost all subsets of leukocytes for which the patients were deficient. Mobilization was transient and followed the pharmacokinetics of the drug, and was dose-dependent throughout the 7 day trial. These results justify testing the drug for chronic safety and hematologic efficacy at the lowest effective dose and eventually for clinical efficacy (wart resolution, reduced incidence of infections). b. The second paper described the first missense mutation, E343K, that causes WHIM syndrome. All previous CXCR4 mutations have been C-tail truncating mutations, including E343X, which terminates at codon 343. We identified three patients with this mutation from a single family, and demonstrated increased signaling in leukocytes stimulated with CXCR4 agonist CXCL12. This finding suggests that all cases of WHIM syndrome due to CXCR4 mutations could be due to loss or mutation of E343. c. The third paper was done in collaboration with Alison McBride of LVD, and reported the tenth known polyomavirus, isolated from a wart sample biopsied from a patient with WHIM syndrome. This opened up new questions regarding the role of polyomavirus in collaboration with HPV in the pathogenesis of warts in WHIM syndrome. 2.) We reported that IL-10 is critical for control of T. cruzi pathogenesis in a mouse model. We identified IL-10 as a target by comparing the clinical, microbiological, immunological and molecular correlates of infection of a sensitive vs a resistant mouse strain to this pathogen. We then tested IL-10 ko mice and found uniform mortality and uncontrolled parasitemia related to T cell expression of IL-10. A non-T cell source was also implicated but not defined. 3.) We found that mouse gut content, both in the large and small bowel, contains potent and effective leukocyte chemotactic and activating factors. The activities appeared to be due to distinct factors in large and small bowel, and in case appeared not to be a protein. In particular, N-formylpeptides, which are produced by all bacteria and are powerful activating factors at specific receptors expressed on phagocytes, do not account for the activity as assessed in Fpr1 ko mice. This work is significant since the gut contains huge numbers of bacteria which produced factors that may interact with the immune system of the gut both in health and disease, particularly in inflammatory bowel disease. Knowledge about these factors could lead to new treatments in IBD. 4.) We defined organ specific immunity in a mouse model of invasive candidiasis in the model, 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. We found that fatal neutrophil-dependent immunopathology is dependent on chemokine receptor Ccr1, which however does not effect candida control. The effect appears to be due to direct action of Ccr1 agonists in the kidney at blood neutrophil Ccr1 which is expressed late in the model, at a time when Ccr1-dependent neutrophil recruitment is observed. The results highlight the delicate balance between the requirement for neutrophils to control Candida, and the harmful effects of uncontrolled neutrophil accumulation and activation on the kidney tissue. The work is significant because it identifies Ccr1 as a potential drug target in invasive candidiasis affecting the kidney.