Animals
Tlr5−/− mice on a C57BL/6 background from Jackson Laboratory were kindly provided by Prof. Huimin Yan (Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China). Specific pathogen-free (SPF) C57BL/6 mice were purchased from Vital River Experimental Animal Company (Beijing, China). In all experiments, genetically modified mice were systematically compared to their age-, and weight-matched WT littermates. All animals were maintained in a temperature-controlled, specific pathogen-free room with a 12-hour light and dark cycle and ad libitum diet (standard laboratory chow and water) in the Experimental Animal Center of the Academy of Military Medical Sciences according to the National Laboratory Animal guidelines (Ministry of Health, P.R. China, 1998). All animal experiments were approved by the Institutional Animal Care and Use Committee of the Beijing Institute of Lifeomics.
Partial Hepatectomy Model
For 2/3 partial hepatectomy studies, male mice between 8 and 10 weeks of age were anesthetized and subjected to a midline laparotomy by aseptic extirpation of the median and left lateral lobes according to the procedure of Higgins and Anderson [27]. Control animals underwent midventral laparotomy without manipulation of the liver (sham surgery). Experiments began between 8:00 AM and 12:00 PM to minimize possible diurnal variations, and mice had access to food and water throughout the testing period. For CBLB502 administration, mice were pretreated with a single dose of CBLB502 (0.2 mg/kg) intraperitoneally 1 hour before partial hepatectomy.
Gene expression datasets
For gene expression analysis, a published dataset was downloaded from GEO repository (GSE95135). Raw data were normalized by the robust multiarray average (RMA) method. We use ExpressVis (http://www.fgvis.com/expressvis/) to visualize the expression of specific genes of interest [28].
Flow cytometry
Liver mononuclear cells (MNCs) were obtained as previously described [23]. Briefly, the liver tissue was dissected, and dissociated in 0.05% type IV collagenase (Sigma-Aldrich, C5138) digestion. Liver specimens were pressed through a 40 μm cell strainer. The single-cell suspension was centrifuged at 50 g for 5 min, and the supernatant was further centrifuged at 320 g for 5 minutes. The pellet containing liver mononuclear cells was resuspended in 10 ml of 30% Percoll (GE, 17089102), then centrifuged at 800 g for 15 minutes without brake. The cell pellet was washed with PBS and then treated with lysis solution (TIANGEN Biotech, RT122) to remove red blood cells. Centrifuge at 400 g for 10 min, resuspend the mononuclear cell pellets in RPMI-1640 medium for Flow cytometric analysis. MNCs was stained with anti-mouse F4/80-FITC (eBioscience, 11-4801-82), anti-mouse CD45.2-PE-Cy7 (Biolegend,109830), anti-mouse Siglec F-APC (Biolegend, 155508), anti-mouse Ly6G-eFluor 450 (eBioscience, 48-9668-82), and anti-mouse CD11b-BV605 (Biolegend, 101257), anti-mouse TLR5-PE (Abcam, ab45119) or anti-mouse IgG2a (Abcam, ab91363). The number of MNCs was counted by 123-count eBeads Counting Beads (eBioscience, 01-1234). Flow cytometric analysis was performed using a BD FACSCalibur instrument (BD Bioscience) and FlowJo software (Tree Star, Ashland, OR).
Histological analysis, immunohistochemistry stainings, and Oil Red O staining
Liver tissues were excised and fixed in 4% paraformaldehyde and then embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) for morphological analysis. Liver tissues were frozen directly in OCT compound for Oil Red O staining. Liver regeneration rate was determined by the BrdU incorporation assay and proliferating cell nuclear antigen (PCNA) immunohistochemistry staining. For BrdU incorporation, 85 μg BrdU (Sigma, B5002) per gram body weight was injected intraperitoneally 2 hours before the mice were sacrificed, and incorporation was visualized immunohistochemically using BrdU immunohistochemistry staining. BrdU and PCNA immunohistochemistry staining were performed following standard protocols (Wuhan Servicebio technology co., LTD), and the percentage of positive cells were analyzed from randomly selected 3 fields of ×200 magnification for each sample. Oil Red O positive areas were quantified in 3 fields per slide under light microscopy (×200). All images of the liver sections were captured using Nikon Digital Sight DS-U3 camera. Image analysis procedures were performed with Image Pro Plus v6.0 (Media Cybernetics, Inc).
Measurements of Cytokines, flagellin concentrations, and aminotransferases
To detect the cytokines in serum, Cytometric bead array (CBA) Mouse TNF Flex Set (BD Biosciences, 558299), Mouse IL-6 Flex Set (BD Biosciences, 558301), Mouse G-CSF Flex Set (BD Biosciences, 560152), Mouse HGF ELISA kit (Abcam, ab223862), Mouse TGFa ELISA kit (Cloud-Clone Corp., SEA123Mu) were used according to manufacturer’s instruction. We used mouse flagellin ELISA kit (Beijing chengzhikewei biotechnology Co., SU-BN28100) for the quantitative determination of serum and liver homogenates flagellin concentrations. Serum ALT and AST were measured according to the IFCC primary reference procedures at Beijing CIC Clinical Laboratory (Beijing, China).
Detection of lipids
Serum lipids quantification was performed with LabAssay™ Cholesterol kit (Wako, 294-65801), LabAssay™ Triglyceride kit (Wako, 290-63701), and LabAssay™ NEFA kit (Wako, 294-63601) following standard methods. Hepatic lipids were assayed using Triglyceride assay kit (Applygen Technologies, E1013), Cholesterol assay kit (Applygen Technologies, E1026), Free fatty acids assay kit (Applygen Technologies, E1000) according to the manufacturer's recommended protocol.
Real-time PCR
Total RNA was extracted by TRIzol (Thermo Fisher Scientific, 15596026) and reverse-transcribed using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, K1622). The cDNAs were amplified with SYBR Green Realtime PCR Master Mix (TOYOBO, QPK-201) by LightCycler 480 real-time PCR detection system (Roche). Relative gene expression was evaluated by the ΔCT method, and Actb was used as an internal control. Genes specific primers were designed by Primer Bank and listed in Table S1 in Supplementary Information.
Western blot
Proteins were extracted from liver specimens homogenized with PBS containing 0.5% Triton X-100 and proteinase inhibitor cocktail (Roche, 04693116001). Liver total protein quantification was performed with the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23227). Protein extracts were denatured in Laemmli sample buffer and separated by SDS-PAGE. Proteins on the gel were transferred to a PVDF membrane and then probed with indicated primary antibodies. Immune complexes on the membrane were detected with HRP-conjugated secondary antibodies and enhanced chemiluminescence reagents (Thermo Fisher Scientific, 34580). The antibodies used were as follows: rabbit monoclonal anti-phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, 3033), rabbit monoclonal anti-NF-κB p65 (Cell Signaling Technology, 8242), rabbit monoclonal anti-phospho-IκBα (Ser32) (Cell Signaling Technology, 2859), mouse monoclonal anti-IκBα (Cell Signaling Technology, 4814), mouse monoclonal anti-phospho-Stat3 (Tyr705) (Cell Signaling Technology, 9145), mouse monoclonal anti-Stat3 (Cell Signaling Technology, 9139), rabbit monoclonal anti-ACTB (ABclonal, AC026). Secondary HRP conjugated antibodies used were goat anti-mouse IgG (ABclonal, AS003), goat anti-rabbit IgG (ABclonal, AS014).
Statistics
Statistics were calculated with GraphPad Prism 7 (GraphPad Software). Results were expressed as means ± standard error of the mean (SEM). A standard two-tailed unpaired Student’s t-test was used to test the significance of differences between two groups. The distribution of variables is tested by the Kolmogorov-Smirnov test. P value <0.05 was considered statistically significant.