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2015财年，我们报告了以下领域的发现：1.动脉粥样硬化; 2.老年痴呆症，3。原发性免疫缺陷疾病WHIM综合征，和4。同种异体移植耐受的机制。 1.）在2016财年，我们开发了第一个CXC趋化因子受体Cxcr 1基因敲除小鼠。 与Cxcr 2不同，Cxcr 1尽管密切相关，但仍然是一个谜。 我们对这种小鼠的开发提供了一个机会来询问它的生物学功能，我们与两个专注于传染病发病机制的小组合作。 白念珠菌致病机制的首次研究是由M.莱昂纳基斯开始时，他在LMI作为一个ACI和完成后，获得了独立的立场，在LCID。 这项工作表明，该受体是重要的控制系统性念珠菌病的控制水平上的中性粒细胞的杀念珠菌机制，而不是在中性粒细胞运输到肾脏的水平。 这一范例得到了第二项合作研究的支持，该研究在肺假单胞菌感染模型中检查了基因敲除小鼠。 总之，这些研究首次将Cxcr 1确定为通过调节中性粒细胞活化的急性感染性疾病发病机制中的关键受体。 2.） 在2016财年，我们报告了一例WHIM综合征患者，这是一种罕见的免疫缺陷疾病，由先前与Waldenstrom巨球蛋白血症相关的新型CXCR 4突变引起。 该突变以与先前描述的WHIM突变相同的方式影响受体。 有趣的是，WHIM患者的受体与Waldenstrom患者的受体发生了相同的移码，但通过不同的遗传机制。 这项工作进一步证明了CXCR 4的C-尾是一个突变的遗传热点。 3.）第三章在2016财年，我们还报道了用巨噬细胞清除剂氯膦酸盐预处理异基因骨髓受体可持久增强造血嵌合体，并促进供体特异性皮肤移植耐受。 供体特异性同种异体移植耐受是移植中的圣杯，如果实现，可以大量增加潜在器官供体的数量，并减少与手术相关的免疫抑制的毒性。 与供者建立造血嵌合体可以促进供者特异性器官移植耐受。 也有人认为干细胞的植入需要空的骨髓龛。 因此，我们通过用氯膦酸盐耗尽受体的骨髓巨噬细胞来动员干细胞，希望增强供体的植入和嵌合，从而增强皮肤移植耐受性。 我们观察到嵌合体在血液、脾脏和骨髓中的持久增强超过200天。 我们已经用这个原理证明的发现，现在转移到其他利基腾出战略。 4.）在2016财年，我们发现，在用克氏锥虫的嗜肌性棘球绦虫菌株控制小鼠急性感染后，寄生虫以低水平持续存在于组织中，与全身性坏死性血管炎的发展相关。感染因子通常被认为是慢性炎症性疾病（包括自身免疫）的潜在触发因素;然而，通常缺乏直接证据。 在我们的研究中，病变发生在许多但不是所有器官和组织中，骨骼肌动脉受到最严重的影响，与肌炎、萎缩、轻瘫/瘫痪和死亡相关。 组织学显示纤维素样血管坏死，骨骼肌肌细胞内罕见无鞭毛体巢，以及主要由炎性单核细胞、F4/80+巨噬细胞和T. cruzi四聚体特异性CD 8 + T淋巴细胞能够产生IFN和TNF，但不能产生IL-17。 T.在感染小鼠的血清中检测到克氏特异性IgG，但抗体沉积和嗜酸性炎症不是病变的特征。 因此，T.小鼠的克氏感染可能是由病原体特异性I型免疫应答驱动的最严重影响骨骼肌的麻痹性全身性坏死性血管炎的特异性感染触发。

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 FY15 we reported discoveries in the following areas: 1. atherosclerosis; 2. Alzheimer's Disese, 3. The primary immunodeficiency disease WHIM syndrome, and 4. mechanisms of allograft tolerance. 1.) In FY16, we developed the first knockout mouse for the CXC chemokine receptor Cxcr1. Unlike Cxcr2, which has been well-studied, Cxcr1, despite being closely related had remained an enigma. Our development of this mouse provided an opportunity to interrogate its biological functions which we did in collaboration with two groups focused on infectious disease pathogenesis. The first study related to Candida albicans pathogenesis was performed by M. Lionakis starting when he was in the LMI as an ACI and completed after obtaining an independent position in LCID. The work showed that the receptor is important for control of systemic candidiasis operating at the level of control of candidicidal mechanisms in neutrophils, not at the level of neutrophil trafficking to kidney. This paradigm was supported by a second collaborative study examining the knockout mice in a model of Pseudomonas infection of the lung. Together, the studies establish Cxcr1 for the first time as a key receptor in acute infectious disease pathogenesis by regulating neutrophil activation. 2.) In FY16, we reported the case of a patient with WHIM syndrome, a rare immunodeficiency disorder, caused by a novel CXCR4 mutation previously associated with Waldenstrom's Macroglobulinemia. The mutation affects the receptor in the same way as previously described WHIM mutations. Interestingly, the same frameshift occurs in the WHIM patient's receptor as in the Waldenstrom's patient's receptor but by distinct genetic mechanisms. This work provides further evidence that the C-tail of CXCR4 is a genetic hotspot for mutation. 3.) In FY16 we also reported that pre-treatment of allogeneic bone marrow recipients with the macrophage depleting agent clodronate durably enhances hematopoietic chimerism and promotes donor-specific skin allograft tolerance. Donor specific allograft tolerance is a holy grail in transplantation that, if achieved, could massively expand the number of potential organ donors and reduce the toxicity from immunosuppression associated with the procedure. It's known that establishing hematopoietic chimerism with the donor can facilitate donor-specific organ allograft tolerance. It's also thought that engraftment of stem cells requires vacant bone marrow niches. We therefore mobilized stem cells by depleting bone marrow macrophages with clodronate in the recipient hoping to enhance donor engraftment and chimerism and hence skin allograft tolerance. We observed durable enhancement of chimerism in blood, spleen and bone marrow out to over 200 days. We have used this proof of principle finding to move on now to other niche vacating strategies. 4.) In FY16, we showed that following control of acute infection of mice with the myotropic Colombiana strain of Trypanosoma cruzi, parasites persisted in tissue at low levels associated with development of systemic necrotizing vasculitis. Infectious agents are often considered potential triggers for chronic inflammatory disease, including autoimmunity; however, direct evidence is usually lacking. In our study, lesions occurred in many but not all organs and tissues, with skeletal muscle arteries most severely affected, associated with myositis, atrophy, paresis/paralysis and death. Histopathology showed fibrinoid vascular necrosis, rare amastigote nests within skeletal muscle myocytes, and massive leukocyte infiltrates composed mainly of inflammatory monocytes, F4/80+ macrophages and T. cruzi tetramer-specific CD8+ T lymphocytes capable of producing IFN and TNF, but not IL-17. T. cruzi-specific IgG was detected in serum from infected mice, but antibody deposits and neutrophilic inflammation were not features of the lesions. Thus, T. cruzi infection of mice may be a specific infectious trigger of paralyzing systemic necrotizing vasculitis most severely affecting skeletal muscle, driven by pathogen-specific Type I immune responses.