Omega-3 Fatty Acid Suppression of Silica-induced Inflammasome Activation in a Novel Alveolar Macrophage Model

摘要 暴露体在自身免疫性和炎症性疾病的发生发展中起着关键作用。在这个职业- Posal，我将阐述饮食中ω-3脂肪酸二十二碳六烯酸在预防 由呼吸性有毒晶状二氧化硅(CSiO)引起的炎症。此前，我们实验室发现， 补充DHA剂量依赖地降低了cSiO_2触发的自体免疫的几个特征的水平。 狼疮易发小鼠模型中的社区活动。在这个模型中，全身炎症发展的一个关键事件是 二氧化硅诱导的肺泡巨噬细胞(AMPH)毒性，包括激活NLRP3炎症。 Masome和释放强大的IL-1细胞因子。目前的文献和我的初步实验表明 DHA及其代谢物，称为专门的前分解介体(SPM)，可能会减弱这种再- 海绵。NLRP3炎症体的激活，这与许多炎症和自身免疫有关 条件，需要一个初始的启动步骤，在此期间，核因子-kB家族转录因子上调炎症因子- 肌小体成分和前IL-1细胞因子。抗炎G蛋白偶联受体(GPCRs) 已发现可与DHA或SPM结合并抑制核因子-kB的激活。然而，分子的体外阐明 在AMPH中，DHA的保护作用受到单个小鼠可获得的细胞数量较少(~105)的限制。 为了解决这个问题，我使用了马克斯·普朗克研究所(MPI)的细胞。MPI细胞是一种自我更新的巨噬细胞系 来源于在添加GM-CSF的培养液中培养的小鼠胎肝，其表型类似于 阿姆。我的初步数据显示，内毒素刺激和cSiO_2治疗后，IL-1细胞因子的释放 通过补充DHA而减弱。我认为补充DHA会增加细胞膜中的DHA MPI细胞，可被释放并代谢成SPM。游离DHA及其SPM可激活抗炎作用- 以自分泌或旁分泌的方式抑制核因子-kB信号，我假设这是一种原发的- 它们通过多种机制预防cSiO_2引起的炎症。在目标1中，磷脂在体内- 测定DHA的比例，观察DHA对IL-1细胞因子释放和核因子-kB活化的影响 评估过了。我还将用化学激动剂、拮抗剂和针对特定GPCRs的siRNA处理细胞，以验证它们的 在DHA信号通路中的作用。在目标2中，将测量添加DHA的细胞的脂质代谢产物谱。我 然后将确定SPM抑制NF-kB激活和IL-1细胞因子释放的程度。最后，化学- 拮抗剂和siRNA将被用来研究所提出的SPM受体的参与。这些前- 巡查将在支持性环境中进行，并提供必要的资源来实现这些目标-- 形容词。我的全面培训计划提供了个人和职业发展，这将有助于我 成为一名成功的独立研究人员。

ABSTRACT The exposome plays a critical role in the development of autoimmune and inflammatory diseases. In this pro- posal, I will address the role of the dietary ω-3 fatty acid docosahexaenoic acid (DHA) in protecting against inflammation induced by the respirable toxicant crystalline silica (cSiO2). Previously, our laboratory found that supplementation with DHA dose-dependently decreased levels of several features of cSiO2-triggered autoim- munity in a lupus-prone mouse model. A key event in the development of systemic inflammation in this model is cSiO2-induced toxicity of the alveolar macrophage (AMph), which involves activation of the NLRP3 inflam- masome and release of potent IL-1 cytokines. The current literature and my preliminary experiments suggest that DHA and its metabolites, known as specialized proresolving mediators (SPMs), may attenuate this re- sponse. Activation of the NLRP3 inflammasome, which is implicated in many inflammatory and autoimmune conditions, requires an initial priming step, during which NF-kB family transcription factors upregulate inflam- masome components and pro-IL-1 cytokines. Anti-inflammatory G-protein coupled receptors (GPCRs) have been identified that bind DHA or SPMs and inhibit NF-kB activation. However, in vitro elucidation of the molecular events of DHA protection in AMph is limited by the low number of cells attainable from a single mouse (~105). To address this, I used Max Planck Institute (MPI) cells. MPI cells are a self-renewing macrophage cell line derived by culturing fetal mouse livers in GM-CSF-supplemented medium and are phenotypically similar to AMph. My preliminary data show that IL-1 cytokine release in response to LPS-priming and cSiO2 treatment is attenuated by DHA supplementation. I propose that DHA supplementation increases DHA in the cell membrane of MPI cells, which can be released and metabolized to SPMs. Free DHA and its SPMs can activate anti-inflam- matory GPCRs in an autocrine or paracrine manner to attenuate NF-kB signaling, which I hypothesize is a pri- mary mechanism by which they protect against cSiO2-induced inflammation. In Aim 1, the phospholipid incorpo- ration of DHA will be measured, and then effects of DHA on IL-1 cytokine release and NF-kB activation will be assessed. I will also treat cells with chemical agonists, antagonists, and siRNA for specific GPCRs to verify their role in DHA signaling. In Aim 2, the lipid metabolite profile of cells supplemented with DHA will be measured. I will then determine the extent to which SPMs suppress NF-kB activation and IL-1 cytokine release. Lastly, chem- ical antagonists and siRNA will be used to investigate the involvement of proposed SPM receptors. These ex- periments will be performed in a supportive environment with the necessary resources to accomplish these ob- jectives. My comprehensive training plan provides personal and professional development, which will assist me in becoming a successful independent researcher.