Department of Allergy and Rheumatology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Immuno-Rheumatology Center, St. Luke's International Hospital, St. Luke's International University, Tokyo, Japan.
Department of Allergy and Rheumatology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Department of Functional Genomics and Immunological Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
J Autoimmun. 2021 May;119:102617. doi: 10.1016/j.jaut.2021.102617. Epub 2021 Mar 4.
Previous gene expression analyses seeking genes specific to antineutrophil cytoplasmic antibody-associated vasculitis (AAV) have been limited due to crude cell separation and the use of microarrays. This study aims to identify AAV-specific gene expression profiles in a way that overcomes those limitations.
Blood samples were collected from 26 AAV patients and 28 healthy controls (HCs). Neutrophils were isolated by negative selection, whereas 19 subsets of peripheral blood mononuclear cells were sorted by fluorescence assisted cell sorting. RNA-sequencing was then conducted for each sample, and iterative weighted gene correlation network analysis (iterativeWGCNA) and random forest were consecutively applied to identify the most influential gene module in distinguishing AAV from HCs. Correlations of the identified module with clinical parameters were evaluated, and the biological role was assessed with hub gene identification and pathway analysis. Particularly, the module's association with neutrophil extracellular trap formation, NETosis, was analyzed. Finally, the module's overlap with GWAS-identified autoimmune disease genes (GADGs) was assessed for validation.
A neutrophil module (Neu_M20) was ranked top in the random forest analysis among 255 modules created by iterativeWGCNA. Neu_M20 correlated with disease activity and neutrophil counts but not with the presence of antineutrophil cytoplasmic antibody. The module comprised pro-inflammatory genes, including those related to NETosis, supported by experimental evidence. The genes in the module significantly overlapped GADGs.
We identified the distinct group of pro-inflammatory genes in neutrophils, which characterize AAV. Further investigations are warranted to confirm our findings as they could serve as novel therapeutic targets.
以前的基因表达分析，由于粗细胞分离和使用微阵列，寻求抗中性粒细胞胞浆抗体相关性血管炎（AAV）特有的基因，受到限制。本研究旨在以克服这些限制的方式鉴定 AAV 特有的基因表达谱。
收集 26 例 AAV 患者和 28 例健康对照者（HCs）的血液样本。通过负选择分离中性粒细胞，而通过荧光辅助细胞分选对 19 个外周血单核细胞亚群进行排序。然后对每个样本进行 RNA 测序，并连续应用迭代加权基因相关网络分析（iterativeWGCNA）和随机森林，以鉴定区分 AAV 和 HCs 的最具影响力的基因模块。评估鉴定模块与临床参数的相关性，并通过鉴定核心基因和通路分析评估其生物学作用。特别地，分析了该模块与中性粒细胞胞外陷阱形成、NETosis 的关联。最后，评估了该模块与全基因组关联研究（GWAS）鉴定的自身免疫疾病基因（GADGs）的重叠，以进行验证。
在 255 个通过 iterativeWGCNA 生成的模块中，随机森林分析中排名最高的是中性粒细胞模块（Neu_M20）。Neu_M20 与疾病活动度和中性粒细胞计数相关，但与抗中性粒细胞胞浆抗体的存在无关。该模块包含促炎基因，包括与 NETosis 相关的基因，实验证据支持。模块中的基因与 GADGs 显著重叠。
我们鉴定了中性粒细胞中独特的促炎基因群，其特征在于 AAV。需要进一步的研究来证实我们的发现，因为它们可以作为新的治疗靶点。
