Jones Rebecca A, Feng Yi, Worth Austen J, Thrasher Adrian J, Burns Siobhan O, Martin Paul
School of Biochemistry, Faculty of Medical and Veterinary Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
J Cell Sci. 2013 Sep 15;126(Pt 18):4077-84. doi: 10.1242/jcs.128728. Epub 2013 Jul 18.
Wiskott-Aldrich syndrome (WAS) and X-linked neutropenia (XLN) are immunodeficiencies in which the function of several haematopoietic cell lineages is perturbed as a result of mutations in the actin regulator WASp. From in vitro cell biology experiments, and biochemical and structural approaches, we know much about the functional domains of WASp and how WASp might regulate the dynamic actin cytoskeleton downstream of activators such as Cdc42, but in vivo experiments are much more challenging. In patients, there is a correlation between clinical disease and genotype, with severe reductions in WASp expression or function associating with complex multilineage immunodeficiency, whereas specific mutations that cause constitutive activation of WASp result in congenital neutropenia. Here, we take advantage of the genetic tractability and translucency of zebrafish larvae to first characterise how a null mutant in zfWASp influences the behaviour of neutrophils and macrophages in response to tissue damage and to clearance of infections. We then use this mutant background to study how leukocyte lineage-specific transgenic replacement with human WASp variants (including normal wild type and point mutations that either fail to bind Cdc42 or cannot be phosphorylated, and a constitutively active mutant equivalent to that seen in XLN patients) alter the capacity for generation of neutrophils, their chemotactic response to wounds and the phagocytic clearance capacity of macrophages. This model provides a unique insight into WASp-related immunodeficiency at both a cellular and whole organism level.
威斯科特-奥尔德里奇综合征(WAS)和X连锁中性粒细胞减少症(XLN)是免疫缺陷疾病,由于肌动蛋白调节蛋白WASp发生突变,导致多个造血细胞谱系的功能受到干扰。通过体外细胞生物学实验、生化和结构方法,我们对WASp的功能结构域以及WASp如何在诸如Cdc42等激活剂下游调节动态肌动蛋白细胞骨架有了很多了解,但体内实验则更具挑战性。在患者中,临床疾病与基因型之间存在相关性,WASp表达或功能的严重降低与复杂的多谱系免疫缺陷相关,而导致WASp组成性激活的特定突变则导致先天性中性粒细胞减少症。在这里,我们利用斑马鱼幼虫的遗传易处理性和透明度,首先表征zfWASp基因敲除突变体如何影响中性粒细胞和巨噬细胞对组织损伤的反应以及感染清除过程中的行为。然后,我们利用这个突变背景来研究用人类WASp变体(包括无法结合Cdc42或不能被磷酸化的正常野生型和点突变,以及与XLN患者中所见的组成性激活突变体等效的突变体)进行白细胞谱系特异性转基因替代如何改变中性粒细胞的生成能力、它们对伤口的趋化反应以及巨噬细胞的吞噬清除能力。该模型在细胞和整个生物体水平上为WASp相关免疫缺陷提供了独特的见解。