Table_8_RNAseq Profiling of Leukocyte Populations in Zebrafish Larvae Reveals a cxcl11 Chemokine Gene as a Marker of Macrophage Polarization During Mycobacterial Infection.XLSX

Macrophages are phagocytic cells from the innate immune system, which forms the first line of host defense against invading pathogens. These highly dynamic immune cells can adopt specific functional phenotypes, with the pro-inflammatory M1 and anti-inflammatory M2 polarization states as the two extremes. Recently, the process of macrophage polarization during inflammation has been visualized by real time imaging in larvae of the zebrafish. This model organism has also become widely used to study macrophage responses to microbial pathogens. To support the increasing use of zebrafish in macrophage biology, we set out to determine the complete transcriptome of zebrafish larval macrophages. We studied the specificity of the macrophage signature compared with other larval immune cells and the macrophage-specific expression changes upon infection. We made use of the well-established mpeg1, mpx, and lck fluorescent reporter lines to sort and sequence the transcriptome of larval macrophages, neutrophils, and lymphoid progenitor cells, respectively. Our results provide a complete dataset of genes expressed in these different immune cell types and highlight their similarities and differences. Major differences between the macrophage and neutrophil signatures were found within the families of proteinases. Furthermore, expression of genes involved in antigen presentation and processing was specifically detected in macrophages, while lymphoid progenitors showed expression of genes involved in macrophage activation. Comparison with datasets of in vitro polarized human macrophages revealed that zebrafish macrophages express a strongly homologous gene set, comprising both M1 and M2 markers. Furthermore, transcriptome analysis of low numbers of macrophages infected by the intracellular pathogen Mycobacterium marinum revealed that infected macrophages change their transcriptomic response by downregulation of M2-associated genes and overexpression of specific M1-associated genes. Among the infection-induced genes, a homolog of the human CXCL11 chemokine gene, cxcl11aa, stood out as the most strongly overexpressed M1 marker. Upregulation of cxcl11aa in Mycobacterium-infected macrophages was found to require the function of Myd88, a critical adaptor molecule in the Toll-like and interleukin 1 receptor pathways that are central to pathogen recognition and activation of the innate immune response. Altogether, our data provide a valuable data mining resource to support infection and inflammation research in the zebrafish model.

巨噬细胞（macrophage）是源自先天免疫系统（innate immune system）的吞噬细胞（phagocytic cell），构成宿主抵御入侵病原体的第一道防御防线。这类具有高度动态性的免疫细胞可呈现特定的功能表型，其中促炎型M1（pro-inflammatory M1）与抗炎型M2（anti-inflammatory M2）极化状态为其两类极端功能表型。近年来，科研人员已通过实时成像技术在斑马鱼（zebrafish）幼体中可视化了炎症过程中的巨噬细胞极化进程。这种模式生物也已被广泛应用于研究巨噬细胞对微生物病原体的应答反应。为满足斑马鱼在巨噬细胞生物学领域日益增长的应用需求，本研究旨在解析斑马鱼幼体巨噬细胞的完整转录组（transcriptome）。本研究对比了巨噬细胞特征基因集与其他幼体免疫细胞的表达特异性，并分析了感染状态下巨噬细胞的特异性表达变化。本研究利用已被广泛验证的mpeg1、mpx与lck荧光报告转基因品系，分别分选并测序了斑马鱼幼体的巨噬细胞、中性粒细胞（neutrophil）与淋巴祖细胞（lymphoid progenitor cell）的转录组。本研究所得结果完整呈现了这几类免疫细胞的基因表达谱，并明确了它们之间的异同点。巨噬细胞与中性粒细胞的特征基因谱之间的显著差异主要集中在蛋白酶家族（proteinase family）范围内。此外，参与抗原呈递与加工过程的基因仅在巨噬细胞中特异性表达；而淋巴祖细胞则表达与巨噬细胞活化相关的基因。与体外极化的人类巨噬细胞转录组数据集进行比对后发现，斑马鱼巨噬细胞表达一套高度同源的基因集，同时包含M1与M2标志物（marker）。此外，对少量被胞内病原体海分枝杆菌（Mycobacterium marinum）感染的巨噬细胞进行转录组分析后发现，受感染的巨噬细胞会通过下调M2相关基因的表达，并过表达特定M1相关基因来改变其转录组应答模式。在感染诱导表达的基因中，人类CXCL11趋化因子（chemokine）基因的同源基因cxcl11aa作为上调幅度最高的M1标志物脱颖而出。研究发现，在海分枝杆菌感染的巨噬细胞中，cxcl11aa的上调表达依赖于Myd88的功能；Myd88是Toll样受体（Toll-like receptor）与白细胞介素1受体（interleukin 1 receptor）通路中的关键衔接分子，这两条通路在病原体识别与先天免疫应答激活过程中发挥核心作用。综上，本研究所得数据为斑马鱼模型中的感染与炎症研究提供了极具价值的数据分析资源。