Xie Yadong, Li Min, Chen Kun, Zhu Haoxiang, Tang Mengyao, Zhou Chun, Zheng Yaoming, Wen Jing, Han Miaomiao, Zhang Jia, Zhao Keqing, Xiao Hui, Li Huabin
ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital, Fudan University, Shanghai, 200031, People's Republic of China.
The Center for Microbes, Development and Health, Institut Pasteur of Shanghai; University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, 200031, People's Republic of China.
J Inflamm Res. 2021 Aug 16;14:3969-3983. doi: 10.2147/JIR.S322875. eCollection 2021.
Necroptosis is an inflammatory cell death associated with a variety of chronic diseases. Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease accompanied by eosinophil and neutrophil infiltration. The role of necroptosis in the pathogenesis of CRSwNP remains elusive.
Cell death, including apoptosis, pyroptosis and necroptosis in control sinonasal mucosa and CRSwNP, were analyzed by immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF) staining for cleaved caspase 3, cleaved gasdermin D and p-MLKL, respectively. Correlations between necroptosis, inflammatory cytokines and neutrophil infiltration were assessed and a possible role of necroptosis in CRSwNP was evaluated. Primary nasal polyp cells (DNPCs) were stimulated with damage-associated molecular patterns (DAMPs) including ATP or IL-1α and their expression of inflammatory cytokines was analyzed using RT-PCR. The expression of TNF-α and IFNs in nasal polyps was measured by ELISA; human monocyte THP-1 cells were treated with TNF-α or IFN-γ and cell death was measured by LDH release.
Necroptosis, rather than apoptosis or pyroptosis, was overtly activated in both eosinophilic and non-eosinophilic CRSwNP as evidenced by the presence of prominent phosphorylation of MLKL compared to controls. The abundance of DAMPs (IL-1α, HMGB1), inflammatory cytokines (IL-6) and chemokines (IL-8, CXCL-1) were all increased especially in non-eosinophilic CRSwNP. The extent of necroptosis was positively correlated with the abundance of DAMPs and cytokines, and neutrophil infiltration in CRSwNP. In DNPCs, ATP and IL-1α induced the expression of IL-8 and CXCL-1. Macrophage was found to be the predominant cell type positive for p-MLKL in CRSwNP. Concomitant treatment with TNF-α and IFN-γ, which were abundantly present in CRSwNP, triggered marked necroptosis in THP-1 cells.
Necroptosis induced by TNF-α and IFN-γ may facilitate the production and release of a myriad of proinflammatory cytokines and entailed neutrophil infiltration to exacerbate inflammation in CRSwNP.
坏死性凋亡是一种与多种慢性疾病相关的炎症性细胞死亡。伴鼻息肉的慢性鼻窦炎（CRSwNP）是一种伴有嗜酸性粒细胞和中性粒细胞浸润的慢性炎症性疾病。坏死性凋亡在CRSwNP发病机制中的作用仍不清楚。
通过免疫印迹、免疫组织化学（IHC）和免疫荧光（IF）染色分别检测对照鼻窦黏膜和CRSwNP中细胞死亡情况，包括凋亡、焦亡和坏死性凋亡，检测裂解的半胱天冬酶3、裂解的gasdermin D和p-MLKL。评估坏死性凋亡、炎性细胞因子与中性粒细胞浸润之间的相关性，并评估坏死性凋亡在CRSwNP中的可能作用。用包括ATP或IL-1α在内的损伤相关分子模式（DAMPs）刺激原代鼻息肉细胞（DNPCs），并使用RT-PCR分析其炎性细胞因子的表达。通过ELISA检测鼻息肉中TNF-α和IFN的表达；用人单核细胞THP-1细胞用TNF-α或IFN-γ处理，并通过LDH释放检测细胞死亡情况。
与对照组相比，MLKL磷酸化显著增加，表明在嗜酸性和非嗜酸性CRSwNP中，坏死性凋亡而非凋亡或焦亡被明显激活。DAMPs（IL-1α、HMGB1）、炎性细胞因子（IL-6）和趋化因子（IL-8、CXCL-1）的丰度均增加，尤其是在非嗜酸性CRSwNP中。坏死性凋亡程度与CRSwNP中DAMPs、细胞因子丰度及中性粒细胞浸润呈正相关。在DNPCs中，ATP和IL-1α诱导IL-8和CXCL-1的表达。发现巨噬细胞是CRSwNP中p-MLKL阳性的主要细胞类型。TNF-α和IFN-γ在CRSwNP中大量存在，联合处理可在THP-1细胞中引发明显的坏死性凋亡。
TNF-α和IFN-γ诱导的坏死性凋亡可能促进多种促炎细胞因子的产生和释放，并导致中性粒细胞浸润，从而加重CRSwNP中的炎症。
