RNA seq analysis from the kidney neutrophils of pycard mutant adult zebrafish in Mycobacterium marinum infection

Inflammasome regulates the host response to intracellular pathogens including mycobacteria. We have previously shown that the course of M. marinum infection in adult zebrafish (Danio rerio) has similar features than the course of tuberculosis in human. Here we have investigated the role of the inflammasome adaptor pycard in M. marinum infection in zebrafish with two CRISPR/Cas9 mutagenesis generated zebrafish line. Adult zebrafish devoid of pycard showed impaired survival and increased bacterial burden in M. marinum infection. Transcriptome analysis with RNA sequencing of a kidney derived neutrophils of M. marinum infected fish homozygous for pycardptu4 mutation revealed differentially expressed genes compared to WT controls. Differentially expressed genes were associated with neutrophil degranulation, haematopoiesis and PI3K signalling are differentially expressed in the pycard deficient neutrophils when compared to wild type controls. Overall design: Identification of differentially expressed genes in Mycobacterium marinum infection in kidney derived neutrophils of zebrafish devoid of inflammasome adaptor pycard at four weeks post infection. Six samples were analyzed, three biological replicates in two groups, fish homozygous for pycardtpu4 mutation and wild type (WT) control.

炎性体（Inflammasome）可调控宿主对包括分枝杆菌（mycobacteria）在内的胞内病原体的免疫应答。我们既往研究证实，成年斑马鱼（Danio rerio）感染海分枝杆菌（M. marinum）的病程与人类结核病的病程特征高度相似。本研究利用两种经CRISPR/Cas9诱变构建的斑马鱼品系，探究了炎性体接头蛋白pycard在斑马鱼海分枝杆菌感染模型中的作用。实验结果显示，缺失pycard的成年斑马鱼在海分枝杆菌感染后存活率显著下降，且体内细菌负荷升高。我们对携带pycardptu4突变纯合子的海分枝杆菌感染斑马鱼的肾脏来源中性粒细胞开展RNA测序（RNA sequencing）转录组分析，相较于野生型（WT）对照，该组样本存在大量差异表达基因。进一步分析表明，与野生型对照相比，pycard缺陷中性粒细胞中的差异表达基因显著富集于中性粒细胞脱颗粒、造血作用及PI3K信号通路相关功能条目。本研究整体实验设计为：鉴定感染后4周的、缺失炎性体接头蛋白pycard的斑马鱼肾脏来源中性粒细胞在海分枝杆菌感染中的差异表达基因。共纳入6份样本进行分析，分为两组各3个生物学重复：pycardtpu4突变纯合子斑马鱼组与野生型（WT）对照组。