High precision-cut liver slice model to study cell-autonomous antiviral defense of hepatocytes within their microenvironment

Abstract

Background & aims: Increased sensitivity towards tumor necrosis factor (TNF)-induced cell death in virus-infected hepatocytes has revealed a so far unrecognized hepatocyte-intrinsic antiviral immune surveillance mechanism, for which no in vitro or ex vivo model is available. We aimed to establish precision-cut liver slices (PCLS) as a model system to study hepatocyte-intrinsic regulation of apoptosis.

Methods: Preparation of PCLS from mouse and human liver tissue was optimized for minimal procedure-associated apoptosis. Functionality of liver cells in PCLS was characterized using extracellular flux analysis to determine mitochondrial respiration, and viral infection with recombinant adenovirus and lymphocytic choriomeningitis virus (LCMV) was used to probe for hepatocyte-intrinsic sensitivity towards apoptosis in PCLS. Apoptosis was detected by immunohistochemical staining for cleaved-caspase 3 and quantified by detection of effector caspase activity in PCLS.

Results: We established an optimized protocol for preparation of PCLS from human and mouse models using agarose-embedding of liver tissue to improve precision cutting and using organ-protective buffer solutions to minimize procedure-associated cell death. PCLS prepared from virus-infected livers showed preserved functional metabolic properties. Importantly, in PCLS from adenovirus- and LCMV-infected livers we detected increased induction of apoptosis after TNF challenge ex vivo.

Conclusion: We conclude that PCLS can be used as model system to ex vivo characterize hepatocyte-intrinsic sensitivity to cell death. This may also enable researchers to characterize human hepatocyte sensitivity to apoptosis in PCLS prepared from patients with acute or chronic liver diseases.

Lay summary: Virus-infected hepatocytes in vivo show an increased sensitivity towards induction of cell death signaling through the TNF receptor. Studying this hepatocyte-intrinsic antiviral immune surveillance mechanism has been hampered by the absence of model systems that reciprocate the in vivo finding of increased apoptosis of virus-infected hepatocytes challenged with TNF. Herein, we report that an optimized protocol for generation of precision-cut liver slices can be used to study this hepatocyte-intrinsic surveillance mechanism ex vivo.

Keywords: IP3, inositol-3-phosphate; LCMV, lymphocytic choriomeningitis virus; PCLS, precision-cut liver slices; PLCg, phospholipase C gamma; ROS, reactive oxygen species; TNF, tumor necrosis factor; TNF-induced apoptosis; anti-viral immunity; precision-cut liver slices.

Graphical abstract

Fig. 1

Absence of cell death in murine and human PCLS. (A) Serum ALT levels in mice after injection of TNF (400 ng/mouse) at d2 after infection with Ad-CMV-GL (5x10⁸ infectious units/mouse). (B) H&E staining of liver sections at 4 h after TNF-injection in Ad-CMV-GL-infected mice, scale bar: 100 μm. (C) Primary mouse hepatocytes were grown to confluence in 2D-culture and were infected with Ad-CMV-GL before challenge with TNF 2 days later; time kinetics of hepatocyte death after TNF challenge was determined by measuring change in electrical impedance of healthy, healthy/TNF challenged, Ad-CMV-GL-infected and Ad-CMV-GL-infected/TNF challenged hepatocytes. (D) H&E staining of murine and human PCLS directly after preparation and after 2 h of incubation at 37°C, scale bar 100 μm. (E) Time kinetics of LDH release from murine PCLS after incubation at 37°C; lysed PCLS as positive control and culture medium alone as negative control. (F) Immunohistochemistry of PCLS for detection of cleaved-caspase 3 to identify apoptotic cells directly after preparation and after 2 hours of incubation at 37°C. (G) Quantification of cleaved-caspase 3-positive cells in human and murine PCLS (≥3,150 hepatocytes analyzed for each parameter) from (F). (A-G) Representative data from at least 3 separate experiments are shown as mean ± SEM. Statistical significance was calculated using unpaired t test, ∗p ≤0.05, ∗∗p ≤0.01 and ∗∗∗p ≤0.001. LDH, lactate dehydrogenase; PCLS, precision-cut liver slices; sALT, serum alanine aminotransferase; TNF, tumor necrosis factor.

Fig. 2

TNF mediates cell death in PCLS from virus-infected liver. (A) In vivo fluorescence imaging day 2 post infection (Ad-CMV-GIRO, 5x10⁸ infectious units/mouse). (B) Fluorescence images of PCLS prepared from murine liver at day 2 post infection with Ad-CMV-GIRO (5x10⁸ infectious units/mouse). (C) Quantification of fluorescence intensity (radiance) from PCLS (B). (D) H&E staining and immunohistochemistry for cleaved-caspase 3 in PCLS prepared from Ad-CMV-GIRO-infected livers or healthy livers. (E) Mitochondrial stress test of PCLS prepared from Ad-CMV-GIRO-infected livers (5x10⁸ infectious units/mouse) or healthy livers. (F) Quantification of caspase 3 activity by luminescence detection assay in PCLS prepared from Ad-CMV-GIRO-infected liver (5x10⁸ infectious units/mouse) at 2 h after ex vivo TNF challenge (20 ng/ml). (G, H) Quantification of caspase 3-activity in PCLS prepared from Ad-CMV-GIRO infected liver (5x10⁸ infectious units/mouse) after incubation with pharmacological inhibitors of ROS (luteolin), IP₃ receptor signaling (xestospongin), PLCg-signaling (edelfosin) and ex vivo TNF challenge (20 ng/ml). (I) Quantification of caspase 3-activity in PCLS prepared from LCMV strain WE-infected livers at 2 h after ex vivo TNF challenge (20 ng/ml and 100 ng/ml). (A-I) Representative data from at least 3 separate experiments are shown as mean ± SEM. Statistical significance was calculated using unpaired t test, ∗p ≤0.05, ∗∗p ≤0.01 and ∗∗∗p ≤0.001. IP₃, inositol-3-phosphate; LCMV, lymphocytic choriomeningitis virus; PCLS, precision-cut liver slices; PLCg, phospholipase C gamma; ROS, reactive oxygen species; TNF, tumor necrosis factor.