The Neutrophil Lineage in Inflammasomopathies

摘要 对低温比林相关周期性综合征(Caps)的研究形成了我们对先天免疫的看法，并导致了 到CAPS和其他NLRP3依赖型炎症性疾病的治疗方法的临床翻译。我们的 初步的体内遗传学数据表明，单核细胞和巨噬细胞在 上限可能被夸大了。相反，仅中性粒细胞谱系就能在帽子中引起致命性的自体炎症。 新生小鼠，这与全身NLRP3激活没有区别。NLRP3的分子调控 与巨噬细胞相比，中性粒细胞的炎性小体激活是不同的，因此本研究将研究 中性粒细胞和单核细胞系小鼠和人细胞中NLRP3激活的生化调节。 我们将研究来自CAPS患者和激活NLRP3突变的小鼠模型的细胞 在中性粒细胞(Nlrp3PMN小鼠)中特异表达。中性粒细胞前体细胞在成功治疗后升高 在CAPS患者和Nlrp3PMN小鼠中，表明Nlrp3PMN小鼠中性粒细胞发育异常， 或降低中性粒细胞系诱导下垂的阈值--这些假说将得到正式检验 在这项研究中。来自纯化的中性粒细胞前体群体的单细胞Western和RNA-Seq数据显示 健康小鼠中性粒细胞和未成熟中性粒细胞的结构性IL-1b表达 这些前体，但不是成熟的中性粒细胞，是CAPS患者器官中IL-1b的主要来源。我们 现在假设这种受发育调节的前IL-1b的表达能够使中性粒细胞 具有NLRP3激活突变的前体细胞独立于信号1或信号释放经处理的IL-1b 2.我们还假设，中性粒细胞的所有器官和组织中都没有成熟的中性粒细胞- 特殊的CAPS小鼠是下睑下垂的结果。这个项目试图定义：(A)器官的差异 单核细胞或中性粒细胞特异性NLRP3激活的小鼠的病理和发病率；(B)细胞固有 NLRP3激活对中性粒细胞分化和寿命的影响；以及 CAPS患者和新生CAPS小鼠的中性粒细胞对规范和非规范激活物的谱系 炎症性小体和下垂；因此，我们的具体目标是：(1)比较器官受累和发病率 单核细胞特异性和中性粒细胞特异性激活NLRP3的小鼠；(2)理解为什么成熟 中性粒细胞特异性CAPS小鼠的新生儿中没有中性粒细胞；以及(3)调查炎症体 诊断为CAPS的患者中性粒细胞系细胞的激活和下垂诱导。鉴定 导致CAPS疾病的关键细胞类型将有助于我们了解其他NLRP3驱动的疾病的发展 炎症性疾病，还可能改善疾病管理，突出新的生物标志物 自体炎症。

Abstract The study of cryopyrin associated periodic syndromes (CAPS) has shaped our view of innate immunity, and led to the clinical translation of therapies for CAPS and other NLRP3-dependent inflammatory diseases. Our preliminary in vivo genetic data now suggest that the assumed central role of monocytes and macrophages in CAPS may be overstated. Rather, the neutrophil lineage alone can drive lethal autoinflammation in CAPS neonatal mice, and this is indistinguishable from systemic NLRP3 activation. The molecular regulation of NLRP3 inflammasome activation is distinct in neutrophils compared to macrophages, so this research will study biochemical regulation of NLRP3 activation in mouse and human cells of the neutrophil and monocyte lineages. We will investigate cells from patients with CAPS, and from mouse models with activating NLRP3 mutations expressed specifically in neutrophils (Nlrp3PMN mice). Neutrophil precursors are elevated in successfully-treated CAPS patients and in Nlrp3PMN mice, suggesting developmental abnormalities of neutrophils in Nlrp3PMN mice, or a reduced threshold for pyroptosis induction in the neutrophil lineage - hypotheses that will be formally tested in this study. Single cell Western and RNA-Seq data from purified neutrophil precursor populations have revealed constitutive IL-1b expression in neutrophil progenitors and immature neutrophils in healthy mice, suggesting that these precursors, but not mature neutrophils, are the dominant source of IL-1b in organs of CAPS patients. We now hypothesize that this developmentally-regulated expression of pro-IL-1b enables neutrophil precursors with NLRP3 activating mutations to release processed IL-1b independent of signal 1 or signal 2. We also hypothesize that the absence of mature neutrophils from all organs and tissues of neutrophil- specific CAPS mice is a consequence of pyroptosis. This project seeks to define: (a) the differences in organ pathology and morbidity of mice with monocyte- or neutrophil-specific NLRP3 activation; (b) the cell-intrinsic effects of NLRP3 activation on neutrophil differentiation and lifespan; and (c) the sensitivity of cells of the neutrophil lineage in CAPS patients and neonatal CAPS mice to canonical and non-canonical activators of the inflammasome and pyroptosis; Our specific aims are therefore to: (1) compare organ involvement and morbidity of mice with monocyte-specific and neutrophil-specific activation of NLRP3; (2) understand why mature neutrophils are absent in neonates of neutrophil-specific CAPS mice; and (3) investigate inflammasome activation and pyroptosis induction in neutrophil lineage cells from patients diagnosed with CAPS. Identification of the key cell types causing disease in CAPS will help us to understand the development of other NLRP3-driven inflammatory diseases, and may also improve disease management and highlight novel biomarkers for autoinflammation.