Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, Frankfurt, Germany.
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine & Pharmacology TMP, Frankfurt, Germany.
Theranostics. 2019 Mar 16;9(7):2003-2016. doi: 10.7150/thno.28057. eCollection 2019.
Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option. To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line and the mouse model of polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved by adenoviral infection and transposon-based gene transfer using hydrodynamic injection. We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers. These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.
细胞毒性 T 淋巴细胞（CTL）的激活导致脓毒症期间的肝损伤，但涉及的机制在很大程度上尚不清楚。了解潜在的原理将允许干扰 CTL 的激活，从而提供新的治疗选择。为了阐明导致 CTL 激活的机制，我们使用 Hepa1-6 细胞系和多微生物脓毒症的小鼠模型，在野生型、髓样特异性 NOX-2、全局 NOX2 和 NOX4 基因敲除小鼠以及它们的存活率方面进行研究。作为最终的研究结果。在这种情况下，我们还确定了肝组织的 mRNA 和蛋白质表达以及肝损伤的临床参数 - 天冬氨酸和丙氨酸氨基转移酶。通过腺病毒感染和基于转座子的基因转移，使用水力注射实现了 PD-L1 在肝细胞中的特异性过表达。我们观察到在脓毒症小鼠模型中 PD-L1 在肝细胞上的下调。腺病毒和基于转座子的基因转移以恢复 PD-L1 的表达，显著提高了存活率并减少了肝损伤的释放，因为 PD-L1 是负调控 T 细胞功能的共受体。在使用重组 PD-L1-Fc 的药理学干预期间观察到了类似的保护作用。N-乙酰半胱氨酸阻断了 PD-L1 的下调，这表明活性氧的参与。这得到了证实，因为我们观察到在假手术中，NOX4 基因敲除小鼠的 PD-L1 表达显著上调，而其在全局和髓系 NOX2 基因敲除小鼠中的表达与野生型动物相当。只有在总 NOX2 基因敲除小鼠中，CLP 后 PD-L1 的表达仍然很高，导致肝损伤标志物的释放明显减少。这些结果表明，与普遍的假设相反，维持肝细胞上的 PD-L1 表达可改善脓毒症期间小鼠的肝损伤和存活率。我们得出结论，给予重组 PD-L1 或抑制 NOX2 活性可能为脓毒症提供新的治疗选择。
