Genetic or pharmacological reduction of cholangiocyte senescence improves inflammation and fibrosis in the Mdr2^-/- mouse

Background & Aims: Cholangiocyte senescence is important in the pathogenesis of primary sclerosing cholangitis (PSC). We found that CDKN2A (p16), a cyclin-dependent kinase inhibitor and mediator of senescence, was increased in cholangiocytes of patients with PSC and from a PSC mouse model (multidrug resistance 2; Mdr2^-/-). Given that recent data suggest that a reduction of senescent cells is beneficial in different diseases, we hypothesised that inhibition of cholangiocyte senescence would ameliorate disease in Mdr2^-/- mice. Methods: We used 2 novel genetic murine models to reduce cholangiocyte senescence: (i) p16^Ink4a apoptosis through targeted activation of caspase (INK-ATTAC)xMdr2^-/-, in which the dimerizing molecule AP20187 promotes selective apoptotic removal of p16-expressing cells; and (ii) mice deficient in both p16 and Mdr2. Mdr2^-/- mice were also treated with fisetin, a flavonoid molecule that selectively kills senescent cells. p16, p21, and inflammatory markers (tumour necrosis factor [TNF]-α, IL-1β, and monocyte chemoattractant protein-1 [MCP-1]) were measured by PCR, and hepatic fibrosis via a hydroxyproline assay and Sirius red staining. Results: AP20187 treatment reduced p16 and p21 expression by ~35% and ~70% (p >0.05), respectively. Expression of inflammatory markers (TNF-α, IL-1β, and MCP-1) decreased (by 60%, 40%, and 60%, respectively), and fibrosis was reduced by ~60% (p >0.05). Similarly, p16^-/-xMdr2^-/- mice exhibited reduced p21 expression (70%), decreased expression of TNF-α, IL-1β (60%), and MCP-1 (65%) and reduced fibrosis (~50%) (p >0.05) compared with Mdr2^-/- mice. Fisetin treatment reduced expression of p16 and p21 (80% and 90%, respectively), TNF-α (50%), IL-1β (50%), MCP-1 (70%), and fibrosis (60%) (p >0.05). Conclusions: Our data support a pathophysiological role of cholangiocyte senescence in the progression of PSC, and that targeted removal of senescent cholangiocytes is a plausible therapeutic approach. Lay summary: Primary sclerosing cholangitis is a fibroinflammatory, incurable biliary disease. We previously reported that biliary epithelial cell senescence (cell-cycle arrest and hypersecretion of profibrotic molecules) is an important phenotype in primary sclerosing cholangitis. Herein, we demonstrate that reducing the number of senescent cholangiocytes leads to a reduction in the expression of inflammatory, fibrotic, and senescence markers associated with the disease.