Regulation of IFN-beta induction by P2X7 purinergic receptor signaling

目标：我的长期研究目标是更深入地了解巨噬细胞对I型IFN产生的调节，巨噬细胞是炎症的关键细胞。作为一名医生兼科学家，我希望这些研究最终有助于改善炎症和自身免疫性疾病的治疗。我近期的职业目标是开发一个可行的独立研究项目。研究项目：在炎症期间，组织损伤的发生导致细胞外嘌呤核苷酸的充分释放，通过嘌呤能受体如P2X7刺激免疫细胞。众所周知，ATP刺激P2X7可增加内毒素（LPS）刺激巨噬细胞产生的炎性细胞因子（如IL-1）。我们发现，LPS和ATP类似物BzATP共同刺激巨噬细胞，大大增强了LPS对IFN-¿的诱导作用。I型干扰素在先天免疫和适应性免疫的各个方面发挥重要作用。由于IFN-¿和嘌呤能受体信号都与细菌脓毒症和骨形态发生有关，因此了解P2X7如何调节IFN-¿的诱导机制至关重要。为了开始解剖P2X7如何发挥其作用的机制，我们提出了一种多管齐下的方法：我们将研究P2X7连接对不同感染刺激（如LPS， dsRNA等）诱导IFN-¿的影响，以确定P2X7增强是否具有LPS特异性。已知P2X7连接可启动多种信号转导途径：我们将分析钙和map -激酶信号通路对BzATP诱导IFN-¿的影响的贡献。最后，我们将通过染色质免疫沉淀研究转录因子对IFN-¿基因启动子的募集，以验证我们的基本假设，即P2X7连接导致转录因子占用ifnb1启动子的增加。P2X7介导的信号通路对转录因子募集的修饰对增强免疫应答和骨生物学具有重要影响。职业发展计划：两个关键组成部分是与Paul Bertics密切合作，以及由指导委员会进行更正式的监督。Paul Bertics在我感兴趣的领域拥有丰富的专业知识，并且在培训初级教师方面有着出色的记录。除了Bertics博士，我的委员会还由另外三位非常支持我的成功的内科科学家组成。研究环境：我通过儿科获得了实验室空间、保护时间和初始启动资金。威斯康星大学医学院和研究生院的专业知识非常广泛。我需要的任何设备或技术援助都可以提供。这里有丰富的论坛供知识交流以及临床和基础科学方面的继续教育。

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.