Department of Otorhinolaryngology-Head and Neck Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; Department of Otorhinolaryngology, The First Affiliated Hospital, Shihezi University, Shihezi 832000, Xinjiang, China.
Department of Otorhinolaryngology-Head and Neck Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Int Immunopharmacol. 2023 Aug;121:110554. doi: 10.1016/j.intimp.2023.110554. Epub 2023 Jun 27.
Previous studies have shown that epithelial-to-mesenchymal transition (EMT) in nasal epithelial cells is critical for tissue remodeling of chronic rhinosinusitis with nasal polyps (CRSwNP). However, the precise mechanism underlying the EMT remains poorly understood. This study aimed to investigate the role of interleukin-4 (IL-4)/signal transducer and activator of transcription 6 (STAT6)/interferon regulatory factor 4 (IRF4) signaling pathway on EMT in eosinophilic CRSwNP.
We performed quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescent staining, and Western blotting to evaluate the expression of STAT6, IRF4, and EMT markers in sinonasal mucosal samples. Effects of IL-4-induced EMT were determined using primary human nasal epithelial cells (hNECs) from patients with eosinophilic CRSwNP. Wound scratch assay, cell morphology, Western blotting, and immunofluorescence cytochemistry were performed to evaluate EMT, and EMT-related markers. Next, human THP-1 monocytic cells were stimulated by phorbolate-12-myristate-13-acetate to differentiate into M0 and were subsequently polarized into M1 with lipopolysaccharide and interferon-γ, M2 with IL-4. The markers of the macrophage phenotype were assessed by Western blotting. The co-culture system was built to explore the interaction between macrophages (THP-1 cells) and hNECs. After co-culture with M2 macrophages, EMT-related markers of primary hNECs were evaluated by immunofluorescence cytochemistry and Western blotting. Enzymelinked immunosorbent assays were used to detect transforming growth factor beta 1 (TGF-β1) in THP-1-derived supernatants.
STAT6 and IRF4 mRNA and protein expression were significantly upregulated in both eosinophilic and noneosinophilic nasal polyps compared with control tissues. The expression of STAT6 and IRF4 in eosinophilic nasal polyps was higher than those in noneosinophilic nasal polyps. STAT6 and IRF4 were not only expressed in epithelial cells but also in macrophages. The number of STAT6CD68 cells and IRF4CD68 cells in eosinophilic nasal polyps was higher than those in noneosinophilic nasal polyps and control tissues. EMT was enhanced in eosinophilic CRSwNP compared to the healthy controls and noneosinophilic CRSwNP. IL-4-stimulated human nasal epithelial cells exhibited EMT characteristics. The hNECs co-cultured with M2 macrophages demonstrated high levels of EMT-related markers. The TGF-β1 level was significantly induced by IL-4 and elevated (M2) rather than control macrophages. The inhibition of STAT6 by AS1517499 reduced the expression of IRF4 in epithelial cells and macrophages and counteracted IL-4-induced EMT in epithelial cells.
In eosinophilic nasal polyps, IL-4 induces STAT6 signaling to upregulate IRF4 expression in epithelial cells and macrophages. IL-4 promotes EMT of hNECs through the STAT6/IRF4 signaling pathway. IL-4-induced M2 macrophages enhanced EMT of hNECs. Inhibition of STAT6 can downregulate the expression of IRF4 and suppress the EMT process, thus providing a new strategy for the treatment of nasal polyps.
先前的研究表明，鼻上皮细胞中的上皮-间充质转化（EMT）对于慢性鼻-鼻窦炎伴鼻息肉（CRSwNP）的组织重塑至关重要。然而，EMT 的精确机制仍知之甚少。本研究旨在探讨白细胞介素 4（IL-4）/信号转导和转录激活因子 6（STAT6）/干扰素调节因子 4（IRF4）信号通路在嗜酸性粒细胞 CRSwNP 中的 EMT 中的作用。
我们通过定量实时聚合酶链反应、免疫组织化学、免疫荧光染色和 Western blot 分析评估了 STAT6、IRF4 和 EMT 标志物在鼻黏膜组织样本中的表达。使用来自嗜酸性粒细胞 CRSwNP 患者的原代人鼻上皮细胞（hNEC）来确定 IL-4 诱导的 EMT。通过划痕实验、细胞形态学、Western blot 和免疫荧光细胞化学评估 EMT 和 EMT 相关标志物。然后，用佛波醇 12-肉豆蔻酸 13-乙酸酯刺激人 THP-1 单核细胞分化为 M0，并用脂多糖和干扰素-γ将其极化为 M1，用 IL-4 将其极化为 M2。通过 Western blot 评估巨噬细胞表型的标志物。建立共培养系统以探索巨噬细胞（THP-1 细胞）和 hNEC 之间的相互作用。与 M2 巨噬细胞共培养后，通过免疫荧光细胞化学和 Western blot 评估原代 hNEC 的 EMT 相关标志物。酶联免疫吸附试验用于检测来自 THP-1 上清液中的转化生长因子β 1（TGF-β1）。
与对照组织相比，嗜酸性和非嗜酸性鼻息肉中 STAT6 和 IRF4 mRNA 和蛋白表达均显著上调。嗜酸性鼻息肉中 STAT6 和 IRF4 的表达高于非嗜酸性鼻息肉。STAT6 和 IRF4 不仅在上皮细胞中表达，也在巨噬细胞中表达。嗜酸性鼻息肉中 STAT6CD68 细胞和 IRF4CD68 细胞的数量高于非嗜酸性鼻息肉和对照组织。与健康对照和非嗜酸性 CRSwNP 相比，嗜酸性 CRSwNP 中 EMT 增强。IL-4 刺激的人鼻上皮细胞表现出 EMT 特征。与 M2 巨噬细胞共培养的 hNEC 显示出高水平的 EMT 相关标志物。TGF-β1 水平由 IL-4 显著诱导并升高（M2）而不是对照巨噬细胞。用 AS1517499 抑制 STAT6 可降低上皮细胞和巨噬细胞中 IRF4 的表达，并拮抗 IL-4 诱导的上皮细胞 EMT。
在嗜酸性鼻息肉中，IL-4 诱导 STAT6 信号通路上调上皮细胞和巨噬细胞中 IRF4 的表达。IL-4 通过 STAT6/IRF4 信号通路促进 hNEC 的 EMT。IL-4 诱导的 M2 巨噬细胞增强了 hNEC 的 EMT。抑制 STAT6 可下调 IRF4 的表达并抑制 EMT 过程，从而为鼻息肉的治疗提供了新策略。
