Sphingosine 1 phosphate receptors 1 and 3 as novel targets in transplantation

描述(由申请者提供)：这份指导研究科学家发展奖(K01)申请书描述了一项职业发展和研究计划，旨在将我定位为树突状细胞(DC)S1P1和S1P3依赖的移植排斥反应免疫机制的独立研究员。移植物功能延迟(DGF)进展为移植中的急性排斥反应是一个重要的临床问题，具有较高的发病率和死亡率，易导致慢性肾脏疾病和移植物丢失甚至死亡。此外，缺血依赖的DGF是移植器官功能的主要决定因素，导致长期的有害影响，包括由于炎症免疫反应的增强而导致的急性和慢性排斥反应发生率的增加。肾脏炎症被公认为急性排斥反应的主要致病因素。作为一名博士后，我的工作最近揭示了肾脏近端小管鞘氨醇-1-磷酸受体-1(S1P1)激活的一个新的作用。肾近端小管S1P1激活保护肾脏免受IRI的影响。此外，在皮肤和心脏移植研究中，缺乏S1P3的DC不能诱导肾脏IRI后的先天免疫反应，缺乏S1P3可以延长移植物的存活时间。此外，VPC01091(S1P1激动剂和S1P3拮抗剂)治疗可延长皮肤移植物存活时间，保护肾脏免受IRI。我们推测，S1P3的激活和S1P1对DC的抑制调节DC的成熟，从而引发炎症，导致DGF和急性排斥反应。在本方案中，我们将阐明：1)利用遗传学方法(使用S1pr3-/-和DC特异性S1PR1-/-小鼠)激活DC S1P1和S1P3在啮齿动物肾移植模型中的作用；2)S1P受体激动剂VPC01091在肾移植中的作用；3)缺乏S1P1和S1P3的DC体外成熟和激活的机制。从这个项目中获得的知识将揭示DC生理学的新方面，并将提供信息，为未来的临床前和临床研究探索VPC01091在移植中的治疗潜力奠定基础。通过这项研究计划，我们将1)熟悉啮齿动物肾脏移植模型，2)利用体内和体外免疫学分析来确定与肾脏疾病相关的移植物存活机制，3)在肾脏损伤的背景下全面了解DC的生理学(从先天/获得性免疫的靶细胞，到DC成熟的机制，到损伤期间的转运模式，再到DC中S1P激动剂和拮抗剂的药理学)，最终目标是建立和资助一个专注于慢性肾脏疾病治疗的独立实验室。这项研究项目的结果将用于准备一项由NIH资助的独立R01申请，该申请将在K01奖的后半部分提交。职业发展计划包括在阿拉巴马大学(UAB)杰出的乔治·F·奥布莱恩中心(核心B)在Anupam Agarwal博士的指导下进行全面的肾移植培训，教授课程工作以使我掌握先进和全面的免疫学知识，这将补充现有的肾脏损伤机制知识。对于这个奖项，我将得到马克·奥库萨博士的指导，他是一位公认的肾脏损伤免疫机制专家。移植专家Peter Lobo博士(顾问)和药理化合物专家Kevin Lynch博士(VPC01091)将在规划、故障排除和解释结果方面提供额外的协助。在成功完成这个培训计划后，我将准备好建立一个独立的研究生涯，探索鞘氨醇受体在移植中的作用，重点是适应性免疫反应以及鞘磷脂受体和激酶在慢性排斥反应中的作用。

DESCRIPTION (provided by applicant): This Mentored Research Scientist Development Award (K01) application describes a career development and research plan designed to position me as an independent investigator in immunological mechanisms of dendritic cell (DC) S1P1 and S1P3 dependent transplant rejection. The main research focus is progression of delayed graft function (DGF) to acute rejection in transplantation, a significant clinical problem associated with high morbidity and mortality, which predisposes individuals to chronic renal disease and loss of graft or even death. Furthermore, ischemia dependent DGF is a major determinant of organ function in transplantation, leading to long-term deleterious effects, including increased rate of acute and chronic rejection as a consequence of enhancement of inflammatory immune response. Kidney inflammation is well-established as a major pathogenic factor in acute rejection. My work as a postdoctoral fellow has recently revealed a novel role of kidney proximal tubule sphingosine-1-phosphate-receptor-1 (S1P1) activation. Kidney proximal tubule S1P1 activation protects kidneys from IRI. In addition, DCs lacking the S1P3 are unable to induce an innate immune response after kidney IRI and lack of S1P3 prolongs graft survival in skin and heart transplantation studies. Additionally, VPC01091 (S1P1 agonist and S1P3 antagonist) treatment prolongs skin graft survival and protects kidneys from IRI. We hypothesize the activation of S1P3 and inhibition of S1P1 on DC regulates DC maturation, which initiates inflammation leading to DGF and acute rejection. In the current proposal, we will elucidate: 1) the role of DC S1P1 and S1P3 activation in rodent kidney transplantation model by using genetic approaches (use of S1pr3-/- and DC specific S1pr1-/- mice), 2) the role of S1P receptor agonist, VPC01091, pharmacological approach in kidney transplantation, and 3) in-vitro mechanisms of DC maturation and activation using DCs deficient of S1P1 and S1P3. The knowledge gained from this project will reveal new aspects of DC physiology and will provide information that can be a foundation for future pre-clinical and clinical studies to explore the therapeutic potential of VPC01091 in transplantation. Through this research plan we will 1) become proficient with rodent kidney transplant model, 2) utilize in-vivo and in-vitro immunological assays to determine the mechanisms of graft survival that are pertinent to kidney disease, and 3) develop a comprehensive understanding of the physiology of DCs, in the context of kidney injury (from target cells of innate/adaptive immunity, to mechanisms of DC maturation, to trafficking patterns during injury, to the pharmacology of an S1P agonist and antagonist in DCs) with the ultimate goal of establishing and funding an independent laboratory focused on cures and treatments for chronic kidney diseases. The results of this research project will be used in the preparation of an independent NIH funded R01 application that will be submitted in the latter part of the K01 award. The career development plan includes thorough training in kidney transplantation under the guidance of Dr. Anupam Agarwal at the outstanding George F. O'Brien Center (Core B) at the University of Alabama (UAB), didactic course work to equip me with advanced and comprehensive knowledge of immunology, which will complement existing knowledge of mechanisms of kidney injury. For this award, I will be mentored by Dr. Mark Okusa, a recognized expert in immune mechanisms of kidney injury. Additional assistance in planning, troubleshooting, and interpreting results will be provided by Dr. Peter Lobo (consultant) for expertise in transplantation and Dr. Kevin Lynch for expertise in pharmacological compounds (VPC01091). After successful completion of this training program, I will emerge well-equipped to establish an independent research career in exploring the role of sphingosine receptors in transplantation with focus on adaptive immune response and role of sphingolipid receptors and kinases in chronic rejection.