Wiesmeier Michael, Gautam Sanjivan, Kirschnek Susanne, Häcker Georg
Institute for Medical Microbiology and Hygiene, Medical Center-University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany.
Spemann Graduate School of Biology and Medicine, University of Freiburg, Freiburg, Germany.
PLoS One. 2016 Dec 12;11(12):e0168055. doi: 10.1371/journal.pone.0168055. eCollection 2016.
Severe congenital neutropenia (SCN) is characterised by a differentiation block in the bone marrow and low neutrophil numbers in the peripheral blood, which correlates with increased risk of bacterial infections. Several underlying gene defects have been identified in SCN patients. Mutations in the neutrophil elastase (ELANE) gene are frequently found in SCN and cyclic neutropenia. Both mislocalization and misfolding of mutant neutrophil elastase protein resulting in ER stress and subsequent induction of the unfolded protein response (UPR) have been proposed to be responsible for neutrophil survival and maturation defects. However, the detailed molecular mechanisms still remain unclear, in part due to the lack of appropriate in vitro and in vivo models. Here we used a system of neutrophil differentiation from immortalised progenitor lines by conditional expression of Hoxb8, permitting the generation of mature near-primary neutrophils in vitro and in vivo. NE-deficient Hoxb8 progenitors were reconstituted with murine and human forms of typical NE mutants representative of SCN and cyclic neutropenia, and differentiation of the cells was analysed in vitro and in vivo. ER stress induction by NE mutations could be recapitulated during neutrophil differentiation in all NE mutant-reconstituted Hoxb8 cells. Despite ER stress induction, no change in survival, maturation or function of differentiating cells expressing either murine or human NE mutants was observed. Further analysis of in vivo differentiation of Hoxb8 cells in a murine model of adoptive transfer did not reveal any defects in survival or differentiation in the mouse. Although the Hoxb8 system has been found to be useful for dissection of defects in neutrophil development, our findings indicate that the use of murine systems for analysis of NE-mutation-associated pathogenesis is complicated by differences between humans and mice in the physiology of granulopoiesis, which may go beyond possible differences in expression and activity of neutrophil elastase itself.
严重先天性中性粒细胞减少症（SCN）的特征是骨髓中的分化阻滞以及外周血中性粒细胞数量减少，这与细菌感染风险增加相关。在SCN患者中已鉴定出几种潜在的基因缺陷。中性粒细胞弹性蛋白酶（ELANE）基因突变在SCN和周期性中性粒细胞减少症中经常被发现。有人提出，突变的中性粒细胞弹性蛋白酶蛋白的错误定位和错误折叠导致内质网应激并随后诱导未折叠蛋白反应（UPR），这是中性粒细胞存活和成熟缺陷的原因。然而，详细的分子机制仍不清楚，部分原因是缺乏合适的体外和体内模型。在这里，我们使用了一种通过条件性表达Hoxb8从不死化祖细胞系分化中性粒细胞的系统，从而能够在体外和体内产生成熟的近原代中性粒细胞。用代表SCN和周期性中性粒细胞减少症的典型NE突变体的小鼠和人类形式重建NE缺陷的Hoxb8祖细胞，并在体外和体内分析细胞的分化。在所有NE突变体重建的Hoxb8细胞的中性粒细胞分化过程中，都可以重现NE突变诱导的内质网应激。尽管诱导了内质网应激，但未观察到表达小鼠或人类NE突变体的分化细胞在存活、成熟或功能方面有任何变化。在过继转移的小鼠模型中对Hoxb8细胞的体内分化进行的进一步分析未发现小鼠在存活或分化方面有任何缺陷。尽管已发现Hoxb8系统有助于剖析中性粒细胞发育缺陷，但我们的研究结果表明，由于人类和小鼠在粒细胞生成生理学方面存在差异，使用小鼠系统分析NE突变相关的发病机制会变得复杂，这种差异可能超出中性粒细胞弹性蛋白酶本身表达和活性的可能差异。
