Regulation of IFN-beta induction by P2X7 purinergic receptor signaling

描述(申请人提供)：目标：我的长期研究目标是更好地了解巨噬细胞对I型干扰素产生的调节，巨噬细胞是炎症的关键参与者。作为一名内科科学家，我希望这些研究最终将有助于改善炎症和自身免疫状况的治疗。我的直接职业目标是开发一个可行的独立研究项目。研究项目：在炎症过程中，组织损伤的发生会导致细胞外的嘌呤核苷酸大量释放，通过P2X7等嘌呤能受体刺激免疫细胞。众所周知，用三磷酸腺苷(ATP)刺激P2X7可以增加内毒素(LPS)刺激的巨噬细胞产生炎性细胞因子(如IL-1)。我们发现，巨噬细胞与内毒素和三磷酸腺苷类似物BzATP的共同刺激大大增强了内毒素对干扰素的诱导。I型干扰素在先天免疫和获得性免疫的各个方面发挥着重要作用。由于干扰素-β和嘌呤能受体信号转导都与细菌性脓毒症和骨形态发生有关，因此了解P2X7调节干扰素-β诱导的机制是至关重要的。为了开始剖析P2X7如何发挥作用的机制，我们提出了一个多管齐下的方法：我们将检测P2X7结扎对不同感染刺激(如内毒素、dsRNA等)诱导的干扰素-β的影响。以确定P2X7增强是否是内毒素特异性的。已知连接P2X7可启动多条信号转导通路：我们将分析钙和MAP-K信号通路在BzATP对干扰素-β诱导效应中的作用。最后，我们将通过染色质免疫沉淀检测转录因子在干扰素基因启动子上的募集，以验证我们的假设，即P2X7连接导致ifnb1启动子转录因子占有率增加。通过P2X7介导的信号调节转录因子的募集，对增强免疫反应和骨生物学具有重要意义。职业发展计划：两个关键部分是与保罗·伯蒂斯的密切合作和由指导委员会进行更正式的监督。保罗·伯蒂斯在我感兴趣的领域拥有丰富的专业知识，在培训初级教员方面有着出色的记录。除了伯蒂斯博士，我的委员会还由另外三位非常支持和成功的内科科学家组成。研究环境：通过儿科，我获得了实验室空间、保护时间和初始启动资金。威斯康星大学通过医学院和研究生院提供的专业知识是巨大的。我需要的任何设备或技术援助都将可用。在临床和基础科学的知识交流和持续教育方面，有大量的论坛。 相关性：巨噬细胞通过分泌细胞因子参与许多炎症过程，包括对感染和自身免疫性疾病的反应。我的研究建议研究被称为“嘌呤”的组织损伤释放的物质是如何调节巨噬细胞产生炎性细胞因子干扰素的。对炎症过程的更多了解可能最终会导致对疾病的更好治疗。

DESCRIPTION (provided by applicant): Goals: My long-term research goal is to gain a greater understanding of the regulation of type I IFN production by macrophages, cells that are key players in inflammation. As a physician-scientist, it is my hope that these studies will eventually contribute to the improved treatment of inflammatory and autoimmune conditions. My immediate career goal is to develop a viable independent research program. Research project: During inflammation, the occurrence of tissue damage leads to sufficient release of extracellular purine nucleotides to stimulate immune cells via purinergic receptors such as P2X7. Stimulation of P2X7 by ATP is well known to increase the production of inflammatory cytokines (e.g. IL-1¿) by macrophages stimulated with endotoxin (LPS). We have found that co-stimulation of macrophages with LPS and the ATP analogue BzATP greatly augments the induction of IFN- ¿ by LPS. Type I IFNs play important roles in diverse aspects of innate and adaptive immunity. As both IFN- ¿ and purinergic receptor signaling have been implicated in bacterial sepsis and bone morphogenesis, it is critical to understand the mechanism of how P2X7 modulates the induction of IFN- ¿. To begin dissecting the mechanism of how P2X7 exerts its effect, a multi-pronged approach is proposed: We will examine the effect of P2X7 ligation on IFN- ¿ induction by different infectious stimuli (e.g. LPS, dsRNA etc.) to determine whether P2X7 enhancement is LPS specific. P2X7 ligation is known to initiate multiple signal transduction pathways: the contribution of calcium and MAP-kinase signaling pathways to the effect of BzATP on IFN- ¿ induction will be analyzed. Finally, we will examine the recruitment of transcription factors to the IFN- ¿ gene promoter by chromatin immunoprecipitation to test our underlying hypothesis that P2X7 ligation leads to increased transcription factor occupancy of the ifnb1 promoter. Modification of transcription factor recruitment by P2X7 mediated signaling has ramifications for the enhancement of immune responses and bone biology. Career development plan: The two key components are close collaboration with Paul Bertics and more formal oversight by a mentoring committee. Paul Bertics has abundant expertise in my area of interest and an excellent track record in training junior faculty. In addition to Dr. Bertics, my committee is composed of three other very supportive and successful physician scientists. Research environment: Through the department of Pediatrics, I have been accorded lab space, protected time and initial start-up funds. The breadth of expertise present at the University of Wisconsin, through the Medical and Graduate schools, is tremendous. Any equipment or technical assistance I should require will be available. There are ample forums for intellectual exchange as well as continuing education in the clinical and basic sciences. RELEVANCE: Macrophages participate in many inflammatory processes including responses to infections and autoimmune diseases by secreting cytokines. My research proposes to study how substances that are released by tissue damage called "purines" regulate the production of the inflammatory cytokine IFN- ¿ by macrophages. Greater understanding of inflammatory processes may ultimately lead to the better treatment of disease.