Abstract：
Primary sclerosing cholangitis (PSC) is an (auto)immune-mediated cholestatic liver disease with a yet unclear etiology. Increasing evidence points to an involvement of neutrophils in chronic liver inflammation and cirrhosis but also liver repair. Here, we investigate the role of the neutrophil extracellular trap (NET) component myeloperoxidase (MPO) and the therapeutic potential of DNase I and of neutrophil elastase (NE) inhibitor GW311616A on disease outcome in the multidrug resistance 2 knockout (Mdr2-/-) mouse, a PSC animal model. Initially, we observed the recruitment of MPO expressing cells and the formation of NETs in liver biopsies of PSC patients and in Mdr2-/- livers. Furthermore, sera of Mdr2-/- mice contained perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA)-like reactivity similar to PSC patient sera. Also, hepatic NE activity was significantly higher in Mdr2-/- mice than in wild type littermates. Flow cytometry analyses revealed that during disease development a highly active neutrophil subpopulation established specifically in the liver of Mdr2-/- mice. However, absence of their MPO activity, as in MPO-deficient Mdr2-/- mice, showed no effect on hepatobiliary disease severity. In contrast, clearance of extracellular DNA by DNase I reduced the frequency of liver-resident neutrophils, plasmacytoid dendritic cells (pDCs) and CD103+ conventional DCs and decreased cholangiocyte injury. Combination of DNase I with a pDC-depleting antibody was additionally hepatocyte-protective. Most importantly, GW311616A, an orally bioavailable inhibitor of human NE, attenuated hepatobiliary injury in a TNFα-dependent manner and damped hyperproliferation of biliary epithelial cells. Further, hepatic immigration and activity of CD11b+ DCs as well as the secretion of IFNγ by hepatic CD4 and CD8 T cells were reduced. Our findings delineate neutrophils as important participants in the immune cell crosstalk that drives cholestatic liver disease and identify NET components as potential therapeutic targets.
摘要：
原发性硬化性胆管炎（PSC）是一种（自身）免疫介导的胆汁淤积性肝病，病因尚不清楚。越来越多的证据表明中性粒细胞参与慢性肝脏炎症和肝硬化以及肝脏修复。这里,我们研究了中性粒细胞胞外诱捕网（NET）成分髓过氧化物酶（MPO）的作用以及DNaseI和中性粒细胞弹性蛋白酶（NE）抑制剂GW311616A对多药耐药2基因敲除（Mdr2-/-）小鼠疾病结局的治疗潜力，PSC动物模型。最初，我们观察到PSC患者和Mdr2-/-肝脏中MPO表达细胞的募集和NETs的形成。此外，Mdr2-/-小鼠的血清含有类似于PSC患者血清的核周抗中性粒细胞胞浆抗体（p-ANCA）样反应性。此外，Mdr2-/-小鼠的肝NE活性明显高于野生型同窝小鼠。流式细胞术分析显示，在疾病发展过程中，在Mdr2-/-小鼠的肝脏中特异性建立了高活性的中性粒细胞亚群。然而,没有他们的MPO活动，与MPO缺陷型Mdr2-/-小鼠一样，对肝胆疾病的严重程度没有影响。相比之下,DNA酶I清除细胞外DNA减少了肝脏驻留的中性粒细胞的频率，浆细胞样树突状细胞（pDCs）和CD103+常规DCs，减少胆管细胞损伤。DNA酶I与pDC消耗抗体的组合另外是肝细胞保护性的。最重要的是,GW311616A,人NE的口服生物可利用性抑制剂，以TNFα依赖性方式减轻肝胆损伤，并抑制胆道上皮细胞的过度增殖。Further,肝CD4和CD8T细胞的肝迁移和CD11bDCs的活性以及IFNγ的分泌减少。我们的发现将中性粒细胞描述为驱动胆汁淤积性肝病的免疫细胞串扰的重要参与者，并将NET成分识别为潜在的治疗靶标。
