Department of Gerontology, the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310002, China.
Department of Cardiology, Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine, Hangzhou 310006, China.
Int Immunopharmacol. 2023 May;118:110010. doi: 10.1016/j.intimp.2023.110010. Epub 2023 Mar 15.
We investigated whether human umbilical vein endothelial cells (HUVECs) under hypoxic conditions can suppress the production of cytokines in Hut-78 cells via the HIF-1α/PD-L1/PD-1 pathway, and the intervention effect of Nivolumab. HUVECs and HuT-78 cells were monocultured or cocultured in a tri-gas incubator with or without Nivolumab pretreatment. Real-time PCR, western blotting, and protein chips were used. Transcriptional regulation of PD-L1 and PD-1 by HIF-1α was analyzed by ChIP-qPCR and luciferase reporter gene assays. Apoptosis was assessed by flow cytometry. In HuT-78 cells, hypoxic monoculture significantly increased the expression of HIF-1α, PD-1, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, IFN-α, and Bax, decreased the expression of Bcl-2, and resulted in increased apoptosis. In comparison to hypoxic monoculture, hypoxic coculture significantly reduced the expression of IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, and IFN-α, as well as Bcl-2, in HuT-78 cells. Meanwhile, Bax expression was significantly increased with elevated apoptosis in HuT-78 cells. However, pretreatment with Nivolumab significantly antagonized the reduction in cytokines and the elevation in apoptosis in HuT-78 cells. Chip-qPCR and luciferase reporter gene assays demonstrated that hypoxia significantly increased the binding of HIF-1α to the upstream regulatory regions of PD-1 at -63 and -66 bp and PD-L1 at -571 bp, promoting their transcription. Therefore, HUVECs under hypoxia can reduce cytokine production and inhibit their own apoptosis in co-culture with HuT-78 cells via the HIF-1α/PD-L1/PD-1 pathway. These findings provide new clues for exploring the combined use of immune checkpoint inhibitors and anti-angiogenic drugs in clinical settings.
我们研究了在缺氧条件下，人脐静脉内皮细胞（HUVEC）是否可以通过 HIF-1α/PD-L1/PD-1 通路抑制 Hut-78 细胞细胞因子的产生，以及 nivolumab 的干预作用。在三气培养箱中进行 HUVEC 和 Hut-78 细胞的单培养或共培养，或进行 nivolumab 预处理。使用实时 PCR、western blot 和蛋白质芯片进行检测。通过 ChIP-qPCR 和荧光素酶报告基因检测分析 HIF-1α 对 PD-L1 和 PD-1 的转录调控。通过流式细胞术评估细胞凋亡。在 HuT-78 细胞中，缺氧单培养显著增加了 HIF-1α、PD-1、IL-2、IL-4、IL-6、IL-8、IL-10、TNF-α、IFN-α 和 Bax 的表达，降低了 Bcl-2 的表达，并导致细胞凋亡增加。与缺氧单培养相比，缺氧共培养显著降低了 HuT-78 细胞中 IL-2、IL-4、IL-6、IL-8、IL-10、TNF-α 和 IFN-α 的表达，以及 Bcl-2 的表达，同时 Bax 的表达显著增加，细胞凋亡也显著增加。然而，nivolumab 的预处理显著拮抗了 HuT-78 细胞中细胞因子的减少和细胞凋亡的增加。Chip-qPCR 和荧光素酶报告基因检测表明，缺氧显著增加了 HIF-1α 与 PD-1 的上游调控区在-63 和-66 bp 及 PD-L1 在-571 bp 处的结合，促进了它们的转录。因此，缺氧条件下的 HUVEC 可以通过 HIF-1α/PD-L1/PD-1 通路减少与 HuT-78 细胞共培养时的细胞因子产生并抑制自身凋亡。这些发现为探索免疫检查点抑制剂与抗血管生成药物在临床中的联合应用提供了新的线索。
