Blockade of neutrophil extracellular trap components ameliorates cholestatic liver disease in Mdr2 (Abcb4) knockout mice

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.