Supplementary Material for: Altered IL-32 Signaling in Abdominal Aortic Aneurysm

<b><i>Introduction and Objective:</i></b> Interleukin (IL)-32 is a pro-inflammatory cytokine not previously studied in relation to abdominal aortic aneurysm (AAA). The aim of this study was to elucidate the expression and localization of IL-32 in AAA. <b><i>Methods:</i></b> Expression and localization of IL-32 in human aortic tissue was studied with immunohistochemical analysis and Western blot (AAA: <i>n</i> = 5; controls: <i>n</i> = 4). ELISA was used to measure IL-32 in human plasma samples (AAA: <i>n</i> = 140; controls: <i>n</i> = 37) and in media from cultured peripheral blood mononuclear cells (PBMCs) from 3 healthy donors. IL-32 mRNA in PBMCs, endothelial cells, aortic smooth muscle cells (SMCs), and aortic tissue samples of AAA (<i>n</i> = 16) and control aortas (<i>n</i> = 9) was measured with qPCR. <b><i>Results:</i></b> IL-32 was predominantly expressed in SMCs and T-cell-rich areas. Highest mRNA expression was observed in the intima/media layer of the AAA. A weaker protein expression was detected in non-aneurysmal aortas. Expression of IL-32 was confirmed in isolated T cells, macrophages, endothelial cells, and SMCs, where expression was also inducible by cytokines such as interferon-γ. There was no difference in IL-32 expression in plasma between patients and controls. <b><i>Conclusion:</i></b> IL-32 signaling is altered locally in AAA and could potentially play an important role in aneurysm development. Further studies using animal models would be helpful to study its potential role in AAA disease.

**引言与研究目的：** 白细胞介素-32（Interleukin-32, IL-32）是一种此前未被关联至腹主动脉瘤（abdominal aortic aneurysm, AAA）的促炎细胞因子。本研究旨在阐明IL-32在腹主动脉瘤中的表达与定位情况。

**方法：** 本研究采用免疫组化分析及蛋白质印迹（Western blot）检测人主动脉组织中IL-32的表达与定位（AAA组：n=5；对照组：n=4）。通过酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）检测人血浆样本（AAA组：n=140；对照组：n=37）以及3名健康供者外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）培养上清液中的IL-32水平。采用实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qPCR）检测PBMCs、内皮细胞、主动脉平滑肌细胞（smooth muscle cells, SMCs）以及16例AAA患者和9例对照主动脉组织样本中的IL-32 mRNA水平。

**结果：** IL-32主要表达于平滑肌细胞及富含T细胞的区域。AAA的内膜/中膜层中IL-32 mRNA表达水平最高。非动脉瘤性主动脉组织中仅检测到较弱的蛋白表达。在分离的T细胞、巨噬细胞、内皮细胞和平滑肌细胞中均证实存在IL-32表达，且干扰素-γ（interferon-γ, IFN-γ）等细胞因子可诱导其表达。患者与对照组血浆中的IL-32表达水平无显著差异。

**结论：** 腹主动脉瘤局部的IL-32信号通路存在异常，其可能在动脉瘤发生发展过程中发挥重要作用。后续可通过动物模型开展相关研究，以进一步明确IL-32在腹主动脉瘤疾病中的潜在作用。