Abstract

As a key virulence factor for persistent colonization, urease B subunit (UreB) is considered to be an ideal vaccine antigen against Helicobacter pylori infection. However, the role and molecular mechanisms of UreB involved in immune microenvironment dysregulation still remain largely unknown. In the present study, we evaluated the effects of UreB on macrophage activation and found that UreB induced PD-L1 accumulation on bone marrow-derived macrophages (BMDMs). Co-culture assays further revealed that UreB-induced PD-L1 expression on BMDMs significantly decreased the proliferation and secretion of cytolytic molecules (granzyme B and perforin) of splenic CD8⁺ T cells isolated from inactivated H. pylori-immunized mice. More importantly, using liquid chromatography–tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation techniques, it has been confirmed that myosin heavy chain 9 (Myh9) is a direct membrane receptor for UreB and is required for PD-L1 up-regulation on BMDMs. Molecular studies further demonstrated that the interaction between UreB and Myh9 decreased GCN2 autophosphorylation and enhanced the intracellular pool of amino acids, leading to the up-regulation of S6K phosphorylation, a commonly used marker for monitoring activation of mTORC1 signaling activity. Furthermore, blocking mTORC1 activation with its inhibitor Temsirolimus reversed the UreB-induced PD-L1 up-regulation and the subsequent inhibitory effects of BMDMs on activation of cytotoxic CD8⁺ T-cell responses. Overall, our data unveil a novel immunosuppressive mechanism of UreB during H. pylori infection, which may provide valuable clues for the optimization of H. pylori vaccine.

中文翻译：

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螺杆菌脲酶通过激活 Myh9 依赖性诱导的 PD-L1 抑制细胞毒性 CD8+ T 细胞反应

摘要

作为持续定植的关键毒力因子，脲酶B亚基（UreB）被认为是对抗幽门螺杆菌感染的理想疫苗抗原。然而，UreB 在免疫微环境失调中的作用和分子机制仍然很大程度上未知。在本研究中，我们评估了 UreB 对巨噬细胞活化的影响，发现 UreB 可诱导 PD-L1 在骨髓源性巨噬细胞 (BMDM) 上的积累。共培养试验进一步表明，UreB 诱导的 BMDMs 上的 PD-L1 表达显着降低了从灭活的幽门螺杆菌中分离出的脾 CD8 ^{+ T 细胞的溶细胞分子（颗粒酶 B 和穿孔素）的增殖和分泌。}-免疫小鼠。更重要的是，使用液相色谱-串联质谱 (LC-MS/MS) 和免疫共沉淀技术，已证实肌球蛋白重链 9 (Myh9) 是 UreB 的直接膜受体，是 PD-L1 up 所必需的- 对 BMDM 的监管。分子研究进一步表明，UreB 和 Myh9 之间的相互作用降低了 GCN2 的自磷酸化并增强了细胞内氨基酸库，导致 S6K 磷酸化的上调，S6K 磷酸化是监测 mTORC1 信号活性激活的常用标志物。此外，用其抑制剂 Temsirolimus 阻断 mTORC1 激活逆转了 UreB 诱导的 PD-L1 上调和随后 BMDMs 对细胞毒性 CD8 ^{+激活的抑制作用}T细胞反应。总体而言，我们的数据揭示了 UreB 在H. pylori感染期间的一种新的免疫抑制机制，这可能为H. pylori疫苗的优化提供有价值的线索。