Lack of FGF21 promotes NASH-HCC transition via hepatocyte-TLR4-IL-17A signaling

Rationale: Hepatocellular carcinoma (HCC) has been increasingly recognized in nonalcoholic steatohepatitis (NASH) patients. Fibroblast growth factor 21 (FGF21) is reported to prevent NASH and delay HCC development. In this study, the effects of FGF21 on NASH progression and NASH-HCC transition and the potential mechanism(s) were investigated. Methods: NASH models and NASH-HCC models were established in FGF21Knockout (KO) mice to evaluate NASH-HCC transition. IL-17A signaling was investigated in the isolated hepatic parenchymal cells, splenocytes, and hepatocyte and HCC cell lines. Results: Lack of FGF21 caused significant up-regulation of the hepatocyte-derived IL-17A via Toll-like receptor 4 (TLR4) and NF-κB signaling. Restoration of FGF21 alleviated the high NAFLD activity score (NAS) and attenuated the TLR4-triggered hepatocyte-IL-17A expression. The HCC nodule number and tumor size were significantly alleviated by treatments of anti-IL-17A antibody. Conclusion: This study revealed a novel anti-inflammatory mechanism of FGF21 via inhibiting the hepatocyte-TLR4-IL-17A signaling in NASH-HCC models. The negative feedback loop on the hepatocyte-TLR4-IL-17A axis could be a potential anti-carcinogenetic mechanism for FGF21 to prevent NASH-HCC transition.

List of antibodies

Supplementary Methods
Oil-Red-O staining Oil Red O staining for lipid accumulation in cells was performed. In brief, after treatment, the cells in 24-well plate were washed with PBS and fixed in 4% buffered formalin for 5 min at room temperature. The cells were stained with Oil Red O dye for 1 h. All the images were reviewed and analyzed under microscope at 20x magnification.

ELISA assay
The protein levels of FGF21 and IL-17A were determined using an ELISA assay kit (R&D Systems, DY3057, Inc. Minneapolis, MN) according to the manufacturer's instructions. In brief, a 96-well plate was coated with 100 μL per well of the diluted Capture Antibody (4 μg/mL) and incubated overnight at room temperature. Next day, 100 μL dilution of protein standards (15.6-1000 pg/mL) and samples were added into the wells and incubated for 2 h at room temperature. After washing, 100 μL of Detection Antibody (25 ng/mL) was applied for 2 h at room temperature and then 100 μL of the working dilution of Streptavidin-HRP (1: 200) was added to each well and incubated for 20 min at room temperature. Substrate Solution was added for 20 min at room temperature, and stopped by Stop Solution. Optical density (OD) was determined using a microplate reader at 450 nm and 540 nm/570 nm for correction. Concentration of the protein was calculated based on standard curve.

Isolation of cells from the tissues of liver and spleen
For hepatocyte and Kupffer cell isolation, liver was perfused at 5 mL per min with PBS containing EGTA (2.5 mM) and then digested with PBS containing Collagen D (3 mg/mL, #121-728-040; Roche), NaCl (66.7 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; 50 mM), and CaCl2 (4.8 mM). The digested liver was dissected and then gently teased with forceps to suspend the cells in the solution. Cell suspension was filtered through 100-μm nylon cell strainer (BD Biosciences, Franklin Lakes, NJ) and centrifuged at 50 g speed for 2 min, and repeated for 3 times to collect hepatocytes for culture. The top aqueous phase was transferred into a new centrifuge tube and filtered through 70-μm nylon cell strainer (BD Biosciences, Franklin Lakes, NJ). The suspension was centrifuged with Percoll™ PLUS (GE Healthcare, Sweden #17-0891-01) at 1350 g for 10 min at 4 °C to collect non-hepatic parenchymal cells including Kupffer cells, sinusoidal endothelial cells, and satellite cells. The collected cells were washed with DMEM supplemented with 10% FBS, and then were seeded into rat tail collagen (Sigma, USA) precoated, 100-mm tissue-culture plates for 30 min, nonadherent cells were removed by aspiration, and fresh medium was added for Kupffer cell culture. For the splenocyte isolation, in brief, a piece of spleen tissue was placed directly into the DMEM in a petri dish and homogenized between the frosted ends of two sterilized slides. The homogenized spleen was then transferred through a 70 µm cell strainer and mounted on a 50 mL tube. The cell strainer was rinsed with 5-10 mL DMEM to collect the cells. The suspended cells were then centrifuged at 800 g for 3 min. The red blood cells were removed by Ammonium-Chloride-Potassium lysis buffer.
Flow Cytometry Flow Cytometry assay was performed in the single-cell suspensions of isolated cells from liver and spleen. In brief, cytokine stimulation was performed using 2 uL/mL eBioscienceTM Cell Stimulation Cocktail (plus protein transport inhibitors) (#00-4975, Invitrogen, USA) for 16 h before cell staining. The cells were then stained with monoclonal antibodies or isotype controls. In brief, the cells were stained with APC-conjugated anti-mouse CD4 (#553051, eBiosciences, USA) for 20 min at 4 °C, followed by incubation with fixation buffer (#00-8222-49, eBiosciences, USA) and permeabilization buffer (#00-8333-56, eBiosciences, USA) according to the manufacturer's instructions. After twice washing with PBS, the cells were stained with FITC-conjugated antimouse IL-17 (#A15377, Life technologies molecular probes, USA) or PE-conjugated anti-mouse Foxp3 (#1946535, eBioscience, USA) for 1 h at room temperature. The cells were resuspended in PBS buffer to run flow cytometry. Flow cytometry data were then collected using a FACSCalibur (BD Pharmingen) and analyzed using FlowJo X software (vX0.7, Tree Star, San Carlos, CA) to evaluate the percentages of Th17 cells and Treg cells.
RNA extraction and real-time-polymerase chain reaction (RT-PCR) Total RNA was extracted using the TRIzol reagent (Invitrogen, CA). First-strand complimentary DNA (cDNA) was synthesized from total RNA according to the kit protocol provided by manufacturer (Promega, Madison, WI). Quantitative PCR was carried out using the ABI 7300 realtime PCR system (Applied Biosystems, Carlsbad, CA). The target mRNA expression was quantified, and β-actin was used as an endogenous reference. For the list of primers, see Table  2 in the supplemental file. The 2 -ΔΔCt method was used to determine gene quantification with βactin used as an endogenous reference gene. Results were expressed as fold change in gene expression compared to the WT-CD mice.

Western blot analysis
The protein levels were semi-quantified by Western blot analysis. In brief, electrophoresis was performed on 12% SDS-PAGE gel and the proteins were transformed to nitrocellulose membrane. The membranes were incubated with the primary antibodies (see Table 1 antibody list in supplemental file) overnight at 4℃ and with secondary antibody for 1 h at room temperature. The antigen-antibody complex was then visualized using ECL kit (Amersham, Piscataway, NJ). The protein bands were quantified by densitometry analysis and protein expression was presented pixel ratio of target protein vs endogenous reference, GAPDH or β-actin.
Immunohistochemistry staining analysis Immunohistochemical staining was carried out on the paraffin-embedded material using the DAKO EnVision+System Kit. In brief, the sections were deparaffinized and hydrated. The slides were washed with a TRIS-buffer, and peroxidase blocking was performed for 5 min. After rewashing, the primary antibodies were applied for 60 min and then incubated with labeled polymer for 30 min at room temperature. The substrate-chromogen solution (diaminobenzidine) was added as a visualization reagent. Digital images were acquired with the Olympus 1×51 microscope (Olympus, Pittsburgh, PA) at 20x magnification using the Olympus DP72 digital camera and the length of scratch-wound was measured via the cellSens Dimention imaging system. The procedure for the computer image analysis was performed, and the acquired color images from the immunohistochemical staining were defined a standard threshold according to the software specification. The computer program then quantified the threshold area represented by color images. Protein expressions were defined by the percentages of threshold area in acquired color.