TNF-α Suppresses Autophagic Flux in Acinar Cells in IgG4-Related Sialadenitis
TNF-α Suppresses Autophagic Flux in Acinar Cells in IgG4-Related Sialadenitis
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TNF-α 抑制 IgG4 相关唾液腺炎中腺泡细胞的自噬流
DOI：
10.1177/0022034519871890
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发表时间：
2019-08-28
影响因子：
7.6
通讯作者：
Yang, H. Y.
中科院分区：
文献类型：
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作者：
Hong, X.;Min, S. N.;Yang, H. Y.
IgG 4相关性涎腺炎（IgG 4-RS）是一种新发现的免疫介导的全身性纤维炎性疾病，其影响唾液腺并导致唾液过少。肿瘤坏死因子-α（TNF-α）是一种重要的促炎细胞因子，参与了几种唾液腺疾病，但其作用和机制有关腺泡细胞损伤的IgG 4-RS是未知的。在这里，我们发现，TNF-α水平显着增加，在血清和下颌下腺（SMG）的患者，血清TNF-α水平呈负相关，唾液流速。IgG 4-RS SMG的超微结构观察显示大的自噬空泡积聚，以及致密的纤维束，分泌颗粒减少，细胞间隙变宽，线粒体肿胀和内质网扩张。SMG中LC 3和p62的表达水平均升高。TNF-α处理导致SMG-C6细胞和培养的人SMG组织中LC 3 II和p62水平升高，但当与巴弗洛霉素A1处理组合时，其水平没有进一步升高。此外，在SMG-C6细胞中转染Ad-mCherry-GFP-LC 3B证实了TNF-α处理后自噬通量的抑制。免疫荧光成像显示，在患者、TNF-α处理的SMG-C6细胞和培养的人SMG中，LC 3和溶酶体标志物LAMP 2的共染色显著减少，表明自噬体-溶酶体融合减少。此外，在体内、离体和体外，原/成熟组织蛋白酶D的比率升高。通过溶酶体pH值和LysoTracker DND-26荧光强度评估，TNF-α似乎也诱导腺泡细胞中溶酶体的异常酸化。此外，TNF-α治疗诱导SMG-C6细胞中转录因子EB（TFEB）重新分布，这与IgG 4-RS患者中观察到的变化一致。TNF-α增加了细胞外信号调节激酶（ERK）1/2的磷酸化，U 0126对ERK 1/2的抑制逆转了TNF-α诱导的TFEB再分布、溶酶体功能障碍和自噬通量抑制。这些结果表明，TNF-α是一个关键的细胞因子相关的腺泡细胞损伤IgG 4-RS通过ERK 1/2介导的自噬通量抑制。
IgG4-related sialadenitis (IgG4-RS) is a newly recognized immune-mediated systemic fibroinflammatory disease that affects salivary glands and leads to hyposalivation. Tumor necrosis factor-alpha (TNF-alpha) is a critical proinflammatory cytokine involved in several salivary gland disorders, but its role and mechanism regarding acinar cell injury in IgG4-RS are unknown. Here, we found that TNF-alpha level was significantly increased in serum and submandibular gland (SMG) of patients and that serum TNF-alpha level was negatively correlated with saliva flow rate. Ultrastructural observations of IgG4-RS SMGs revealed accumulation of large autophagic vacuoles, as well as dense fibrous bundles, decreased secretory granules, widened intercellular spaces, swollen mitochondria, and expanded endoplasmic reticulum. Expression levels of LC3 and p62 were both increased in patients' SMGs. TNF-alpha treatment led to elevated levels of LC3II and p62 in both SMG-C6 cells and cultured human SMG tissues but did not further increase their levels when combined with bafilomycin A1 treatment. Moreover, transfection of Ad-mCherry-GFP-LC3B in SMG-C6 cells confirmed the suppression of autophagic flux after TNF-alpha treatment. Immunofluorescence imaging revealed that costaining of LC3 and the lysosomal marker LAMP2 was significantly decreased in patients, TNF-alpha-treated SMG-C6 cells, and cultured human SMGs, indicating a reduction in autophagosome-lysosome fusion. Furthermore, the ratio of pro/mature cathepsin D was elevated in vivo, ex vivo, and in vitro. TNF-alpha also appeared to induce abnormal acidification of lysosomes in acinar cells, as assessed by lysosomal pH and LysoTracker DND-26 fluorescence intensity. In addition, TNF-alpha treatment induced transcription factor EB (TFEB) redistribution in SMG-C6 cells, which was consistent with the changes observed in IgG4-RS patients. TNF-alpha increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, and inhibition of ERK1/2 by U0126 reversed TNF-alpha-induced TFEB redistribution, lysosomal dysfunction, and autophagic flux suppression. These findings suggest that TNF-alpha is a key cytokine related to acinar cell injury in IgG4-RS through ERK1/2-mediated autophagic flux suppression.