Supplementary Material for: Downregulation of <b><i>ITM2A</i></b> Gene Expression in Macrophages of Patients with Ankylosing Spondylitis

<b><i>Objectives:</i></b> Ankylosing spondylitis (AS) is a rheumatic disorder that is mostly determined by genetic and environmental factors. Given the known importance of macrophage in AS pathogenesis, we investigated the transcriptional profile of macrophage cells in the disease. <b><i>Methods and Results:</i></b> Two approaches of differential expression and subsequently, weighted gene co-expression network analysis was utilized to analyze a publicly available microarray dataset of macrophages. Integral membrane protein 2A (<i>ITM2A</i>) was among the most significant genes with a decreased trend in the common results of both methods. In order to confirm the finding, the expression of <i>ITM2A</i> was evaluated in monocyte-derived (M2-like) and M1 macrophages obtained from 14 AS patients and 14 controls. Macrophages were differentiated from whole-blood separated monocytes by 7 days incubating with macrophage colony-stimulating factor and then macrophages specific markers were verified with the flow cytometer. M1 polarization was induced by IFN-γ and lipopolysaccharide. Finally, relative gene expression analysis by real-time polymerase chain reaction revealed a significant downregulation of the <i>ITM2A</i> gene in both M2 like and M1 macrophages of the AS group compared to the control. <b><i>Conclusion:</i></b> Since <i>ITM2A</i> plays a critical role in osteo- and chondrogenic cellular differentiation, our finding may provide new insights into AS pathogenesis.

<b><i>研究目的：</i></b> 强直性脊柱炎（Ankylosing spondylitis, AS）是一类主要由遗传与环境因素共同决定的风湿性疾病。鉴于巨噬细胞（macrophage）在AS发病机制中的已知重要作用，本研究针对该疾病中巨噬细胞的转录谱展开探究。<b><i>方法与结果：</i></b> 本研究采用差异表达分析（differential expression analysis）及后续的加权基因共表达网络分析（weighted gene co-expression network analysis）两种方法，对公开可用的巨噬细胞微阵列数据集进行分析。整合膜蛋白2A（<i>ITM2A</i>）是两种方法共同分析结果中最具显著性的基因之一，在AS患者样本中呈现表达下调趋势。为验证该发现，本研究采集14例AS患者与14例健康对照的样本，对其单核细胞衍生的（M2样）及M1型巨噬细胞中<i>ITM2A</i>的表达水平进行检测。通过将全血分离得到的单核细胞与巨噬细胞集落刺激因子（macrophage colony-stimulating factor）共培养7天，诱导分化为巨噬细胞，并通过流式细胞仪（flow cytometer）验证巨噬细胞特异性标志物的表达。随后采用干扰素γ（Interferon-γ, IFN-γ）与脂多糖（lipopolysaccharide, LPS）诱导M1型巨噬细胞极化。最终通过实时荧光定量聚合酶链反应（real-time polymerase chain reaction）进行相对基因表达分析，结果显示：与健康对照组相比，AS组的M2样及M1型巨噬细胞中<i>ITM2A</i>基因均呈现显著下调。<b><i>结论：</i></b> 鉴于<i>ITM2A</i>在骨及软骨细胞分化过程中发挥关键作用，本研究结果可为AS的发病机制研究提供全新视角。