Cytosolic nucleic acid sensors of the innate immune system promote liver regeneration after partial hepatectomy

Stimulation of cytosolic nucleic acid sensors of innate immunity by pathogen-derived nucleic acids is important for antimicrobial defence, but stimulation through self-derived nucleic acids may contribute to autoinflammation and cancer. DNA sensing in the cytosol requires the stimulator of interferon genes (STING), while cytosolic RNA sensors use mitochondrial antiviral-signalling protein (MAVS). In a murine model of two-thirds hepatectomy, combined deficiency of MAVS and STING resulted in strongly impaired hepatocyte proliferation and delayed recovery of liver mass. Whereas lack of MAVS and STING did not influence upregulation of the G1-phase cyclins D1 and E1, it substantially reduced the hyperphosphorylation of retinoblastoma protein, attenuated the activation of cyclin-dependent kinase (CDK)-2, delayed upregulation of CDK1 and cyclins A2 and B1, and impaired S-phase entry of hepatocytes. Mechanistically, lack of cytosolic nucleic acid sensors strongly upregulated the anti-proliferative mediators TGF-β2 and activin A, which was associated with an increased expression of the cell cycle inhibitors p15 and p21. Partial hepatectomy was followed by the release of exosomes with abundant nucleic acid cargo, which may provide ligands for the MAVS and STING pathways. Together, these findings identify a previously unrecognised function of cytosolic nucleic acid sensors of innate immunity for promoting liver regeneration.

Bone marrow-derived macrophages from wildtype and DKO mice were generated by culture of bone marrow cells in RPMI1640 medium containing 10 % fetal calf serum and 15 % L cell-conditioned medium as a source of M-CSF for at least 7 d.
To stimulate cytosolic DNA sensors, macrophages were incubated with 3 μg/ml of the immunostimulatory DNA VACV-70 that was complexed with the transfection reagent LyoVec™ to facilitate its uptake (Invivogen, Toulouse, France).
After 16 h of stimulation, CXCL10 levels were measured in culture supernatants using the Mouse CXCL10 Quantikine ELISA Kit (R&D Systems, Minneapolis, USA) according to manufacturer's instructions.

Supplemental Figure S2
Legend: Liver sections of untreated wildtype and DKO mice (n=6) were stained with hematoxylin/eosin and mounted with Eukitt mounting medium (Merck, Darmstadt, Germany). Stained sections were imaged using an Axiolap microscope attached to an AxioCam MRc5 camera, an EC Plan-Neofluar 10x/0.3 NA and an Achroplan 20x/0.45 NA objective and analyzed using the Axiovision software (all Carl Zeiss Microscopy, Jena, Germany).
Representative images of wildtype and DKO mice are depicted. Scale bars indicate 50µm.

GAPDH (36kD)
Legend: Areas of the gel shown in Figure 2B are marked in red.
The membrane was probed first with anti-p-STAT3 and subsequently with anti-GAPDH antibodies. Predicted molecular weights for p-STAT3 and GAPDH are indicated. Full length gels for Figure 2C STAT3 (88kD) GAPDH (36kD)

Legend:
Areas of the gel shown in Figure 2C are marked in red.
The membrane was probed first with anti-STAT3 and subsequently with anti-GAPDH antibodies. Predicted molecular weights for STAT3 and GAPDH are indicated. Areas of the gel shown in Figure 4A are marked in red.
The membrane was probed first with anti-cyclin D1 and subsequently with anti-Tubulin antibodies. Predicted molecular weights for cyclin D1 and β-tubulin are indicated.

GAPDH (36kD)
Legend: Areas of the gel shown in Figure 4A are marked in red.
The membrane was probed first with anti-GAPDH and subsequently with anti-cyclin E1 antibodies. Predicted molecular weights for cyclin E1 and GAPDH are indicated. Areas of the gel shown in Figure 4A are marked in red.
The membrane was probed first with anti-p-RB and subsequently with GAPDH antibodies. Predicted molecular weights for p-RB and GAPDH are indicated. Areas of the gel shown in Figure 4A are marked in red.
The membrane was probed first with anti-p-CDK2 and subsequently with anti-Tubulin antibodies. Predicted molecular weights for p-CDK2 and β-tubulin are indicated. Areas of the gel shown in Figure 4A are marked in red.