C57/BL6, Par2KO (CD45.2), and CD45.1 mice (B6.Cg-F2rl1tm1Mslb/J and B6.SJL-Ptprca Pepcb/BoyJ, from Jackson labs, strains # 004993 and #002014, respectively, Bar Harbor, ME, USA) were under pathogen-free conditions in the animal facility of the Azrieli Faculty of Medicine, Bar-Ilan University, Safed, Israel. All animal experiments were conducted according to the institutional animal ethical committee guidelines (Permit Number: 91-11-2017), which conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Eighth edition 2011). The animals were maintained at the institutional Experimental Surgical Unit, fed on a normal rodent chow diet, with tap water ad libitum. The mice were housed at a constant temperature and relative humidity under a regular light/dark schedule (12:12). Males and females were used equally in all experiments.
Hepatitis induction administration
WT and Par2KO mice were injected with a single IV injection of 10 mg/kg ConA (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS, Biological Industries, Beit-Haemek, Israel). Mice were sacrificed 1 or 14 days after injection (3–4 mice were in each group). For high-dose ConA treatment, WT and Par2KO mice were injected with a single IV injection of 15mg/kg ConA. In the high-dose experiment, mice were sacrificed 6 h after injection. Blood samples were taken, and the livers were harvested and analyzed by histochemical and immunohistochemical staining (7–12 mice were in each group).
Direct toxicity model
WT and Par2KO mice were injected with a single IP injection of 1 ml/kg (1.594 g/kg) CCl4 (Sigma-Aldrich, St. Louis, MO, USA, 10% solution in olive oil (Yad-Mordechai, Israel). Mice were sacrificed 1 day or 7 days after injection. Blood samples were taken, and the livers were harvested and analyzed by histochemical staining (3–7 mice were in each group).
Liver enzyme analysis
Serum from experimental mice was collected and analyzed by a VetScan VS2 (Abaxis Veterinary Diagnostics, Union City, CA, USA) at the Sanford Burnham Prebys Medical Discovery Institute animal core facility (La Jolla, CA, USA).
Immuno and histochemical staining
The livers were fixed in formaldehyde (4% in PBS, Santa Cruz Biotechnology, Dallas, TX, USA). The livers were embedded in paraffin, sectioned into 5μm using a microtome, and loaded onto microscope glass slides at the Nahariya Galilee Medical Center’s research unit. Hematoxylin and eosin (H&E) as well as Picro-Sirius Red staining were performed. After de-paraffinization and re-hydration, slides were rinsed in distilled water and stained for nuclei with Weigert’s hematoxylin solution for 5 min. Then, the slides were placed in Picro-Sirius red stain for 60 min, then rinsed twice in 0.5% of the acetic acid solution, followed by dehydration quickly through 3 changes to absolute ethanol. The slides were scanned at a magnification of 20×, and images were acquired by the Axio Scan.Z1 microscope (Zeiss, Jena, Germany).
The slides were washed three times with PBS and treated with 0.3% Triton X-100 (MP Biomedicals, Solon, OH, USA) for 15 min, then washed with PBS for 10 min. The slides were incubated in a blocking solution with 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA, USA) for 50 min at room temperature (RT). The slides were incubated with antisera specific for Par2 (1/400, goat, sc-8207, Santa Cruz Biotechnology). HRP-conjugated secondary antibodies (Jackson ImmunoResearch) were used to visually label Par2 localization (DAKO, Carpinteria, CA, USA). The slides were scanned at a magnification of 20× using the Aperio Scanscope FL system (Aperio Technologies Inc., Vista, CA, USA).
The slides were incubated overnight at 4° C with primary antibodies for Par2 (1/200, mouse, Thermos Fisher Scientific, San Diego, CA, USA), CD45 (1/200, rat, Novus Biological, Centennial, CO 80112, USA), and albumin (1/100, gout, R&D Systems Inc., Minneapolis, MN, USA). Secondary antibodies were labeled with Alexa Fluor 488, Alexa Fluor 647, and Rhodamine Red (all from Jackson ImmunoResearch). Nuclei were visualized with DAPI Fluoromount-GTM (Bar Naor Ltd, Ramat Gan, Israel). The slides were imaged using Microscope Axio Imager M2 (Zeiss).
Cell death identification—TUNEL staining
To visualize cell death, TUNEL staining was performed with TUNEL Andy FlouTM 594 Apoptosis detection kit (ABP Biosciences, Beltsville, MD, USA) as described in their protocol. The slides were scanned at a magnification of 20×, and images were acquired by the Axio Scan.Z1 microscope (Zeiss). For analysis, rectangles of 1,000,000μm2 were analyzed by ImageJ for TUNEL positive area (594nm).
Quantification of liver damage
For the ConA experiments, selected areas of the slides were chosen for figures using Aperio Imagescope (version 12 Aperio Technologies Inc.). For analysis, the slide areas were selected and analyzed using the web-based Image Scope viewer. Nuclear density was measured by creating a rectangle of 100μm2 surrounding Par2 positive or negative areas. Necrosis was identified as a lack of nuclear staining in H&E (thus creating pink-distinctive staining).
For the CCl4 experiments, the liver damage was measured using the ZEN software (Zeiss). Damage was measured as the number of fat vacuoles with an area of 25,000μm2 (day 1) or as the percentage area of necrotized lesions in a macroscopic picture (day 7).
Irradiation and bone marrow chimeras
To generate WT hosts recipients of Par2KO BM, 6-week-old WT (CD45.1), mice were lethally irradiated (1000 Rad) in their cages at the Rivka Ziv’s hospital radiology unit (Safed, Israel) and reconstituted with BM isolated from 9-week-old Par2KO donor mice (CD45.2). To generate Par2KO hosts of WT BM, 6-week-old Par2KO mice (CD45.2) were irradiated and reconstituted with BM isolated from 9-week-old WT donor mice (CD45.1) as described above.
As controls, CD45.1 WT host recipients were reconstituted with BM isolated from CD45.2 WT donors as described above. Donor mice were sacrificed by isoflurane overdose, and BM cells were collected from femurs and tibias. BM cells were pooled together and centrifuged at 600 G for 10 min. The supernatant was discarded, and the pellet was resuspended in saline calculated as 100μl per recipient mouse [=100*(n+2)]. Four hours post-irradiation the recipient mice were reconstituted with donor BM by IV injections. Mice were treated with antibiotics [3.3ml of Septrin (40mg/ml sulfamethoxazole and 8mg/ml trimethoprim, Teva Pharmaceutical Industries Ltd., Petah Tikva, Israel) in 250ml drinking water for 3 weeks]. FACS analysis was conducted for the BM replacement validation: 3 weeks after BM replacement, the blood samples were taken from the mice cheek, 50μl blood from each mouse was washed in 1ml PBS/2%FBS (Biological Industries) and centrifuged at 1200 rpm for 5 min. Antibodies were added according to table S1.
Cells were incubated for 40 min in RT. One milliliter of red blood cells (RBC) lysis buffer (Biological Industries) was added, then tubes were incubated for 10 min in RT and were washed in 1ml PBS/2%FBS, centrifuged at 1200 rpm for 5 min. 150ml of FBS was added, and the contents were poured into the FACS tubes and loaded into the Gallios FACS analyzer (Beckman Coulter Life Sciences, Indianapolis, IN, USA). CD4 and CD8 T cells were gated on CD45.1 for WT donor into Par2KO and on CD45.2 for the reciprocal BM replacement. Three weeks post-BM reconstitution, hepatitis induction administration for the two injury models was carried out as described before.
T cell depletion
WT and Par2KO mice were IP injected with 500μg of anti-CD4 and 500μg of anti-CD8 antibodies (Bio X Cell, Lebanon, NH, USA), 2 days prior and 1 day prior to ConA treatment. FACS analysis was conducted to validate the depletion of CD8 and CD4 T cells, as described above in the BM validation section.
Isolation and characterization of liver immune cells
The enzymatic digestion method was applied for the isolation of intrahepatic lymphocytes by modifying a previously described protocol [57. ]. Perfusion buffer (PB) was prepared by diluting 40ml of perfusion buffer concentrate [500ml PBC= 3.55 M NaCl, 168mM KCl (Sigma-Aldrich), 240mM HEPES (Biological Industries), 150mM NaOH (Sigma-Aldrich), in distilled deionized H2O] into 960-mL ultrapure H2O. A pre-warmed syringe was filled with 50ml of PB at 37°C.
Fifty milliliters of dissociation buffer was prepared by 49.5ml PB supplemented with 0.5ml 476mM CaCl2 (Sigma-Aldrich) and 3600U Collagenase Type IV (Worthington Biochemical Corporation, Lakewood, NJ, USA). Pre-warmed syringe was filled with 50mL of dissociation buffer at 37°C.
Mice were euthanized by isoflurane overdose. Suprahepatic inferior vena cava (IVC) was clamped to maintain localized perfusion, and a 23-G needle attached to a syringe filled with pre-warmed PB was gently inserted into the subhepatic IVC. The portal vein was cut to allow PB and blood flow through the liver. The liver was perfused until blood was no longer visible. PB syringe was replaced with the pre-warmed dissociation buffer syringe, and the liver was perfused until it was fully digested. The liver was carefully removed from the mouse to a 10ml ice-cold DMEM (Biological Industries). Then, the Petri dish was gently dispersed using forceps. Saturated DMEM was passed through a 70μm cell strainer (Jet Biofil, Guangzhou, China) and attached to a 50ml collection tube. Cell suspension was centrifuged at 50G for 2 min at 4°C, and the supernatant was collected. This step was repeated 2 more times. Then, the supernatant was centrifuged at 300G for 10 min at 4°C. The cell pellet was washed in PBS/2%FBS. Samples were incubated with FcR Blocking Reagent (Miltenyi Biotec, Auburn, CA, USA) for 10 min at 4°C before being stained with monoclonal antibodies for an additional 20 min at 4°C according to table S1. Cells were washed in 1ml PBS/2%FBS and centrifuged at 1200 rpm for 5 min. 150μl PBS/2%FBS was added, and the contents were transferred into the FACS tubes (1μl of each antibody was added to 1×106 cells in 100μl PBS/2%FBS). Cells were analyzed on a Gallios flow cytometer, and at least 20,000 gated events were analyzed using Kaluza software (Beckman Coulter).
Statistical analysis was carried out using GraphPad Prism 5.0, evaluating the differences between different groups. All data were presented as mean ± SEM. Comparisons between groups for statistical significance were performed with Student’s T test or Mann–Whitney test. Survival curves among groups were assessed by Mantel-Cox log-rank test. Results were considered significant difference at *P < 0.05, **P < 0.01, ***P < 0.005.