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

Abstract

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.

Keywords: ALP, alkaline phosphatase; AP, AP20187; Apoptosis resistance; BCL2, B cell lymphoma 2; Bcl-xL, B-cell lymphoma-extra large; Biliary epithelial cell; CCA, cholangiocarcinoma; CKI, cyclin-dependent kinase inhibitor; Cellular senescence; Cholestatic liver disease; Col.1A, collagen 1A; D, dasatinib; EVs, extracellular vesicles; FKBP-Casp8, FK506-binding-protein-caspase 8; IF, immunofluorescence; INK-ATTAC, p16Ink4a apoptosis through targeted activation of caspase; IR, irradiation; MCL1, myeloid cell leukemia 1; MCP-1, monocyte chemoattractant protein-1; MMP, matrix metalloproteinase; NHC, normal human cholangiocyte; PSC, primary sclerosing cholangitis; Primary sclerosing cholangitis; Q, quercetin; RT, reverse transcription; SA-β-gal, senescence-associated β-gal; SASP, senescence-associated secretory phenotype; Senescence-associated secretory phenotype; Senolytics; TNF, tumour necrosis factor; WT, wild-type; mdr2, multidrug-resistance 2; qPCR, quantitative PCR; α-SMA, α-smooth muscle actin; β-Gal, β-galactosidase.