**1. Introduction**

Neutrophils are the most abundant white blood cells and among the first cells recruited to an inflammatory site, thus mediating the early responses to tissue injury [1]. Neutrophil activation is characterized by neutrophil extracellular trap (NET) formation (NETosis) [2,3], granule enzymes myeloperoxidase (MPO) release from lysosome (azurophilic granules) [4] and ROS burst [5], further contributes to inflammation-associated damage in injured tissue. NETosis was first described in 2004 as highly decondensed chromatin structures, which was associated with citrullination of histone H3 [6,7]. NETosis has been found in response to various stimuli such as LPS, damage-associated molecular patterns (DAMPs), and PMA [6,8]. Recent studies have implicated that neutrophils play an essential role in the control of sterile inflammatory diseases, which are also characterized by a sustained influx of neutrophils and persistent NET release, and contribute to various injury processes [9,10]. For instance, in chronic obstructive pulmonary disease and cystic fibrosis, neutrophils and NETs contribute to chronic inflammatory and lung tissue damage [11,12]. Peptidylarginine deiminase (PAD) inhibition reduces NETosis and protects against lupus-related vasculature, kidney and skin injury in various

lupus models [13]. In pyogenic arthritis, pyoderma gangrenosum and acne syndrome, an imbalance of NET formation and degradation are detected that enhances the half-life of these structures in vivo and promotes inflammation [14]. Especially, the important role of neutrophils has been identified in acute liver injury. In liver ischemia/reperfusion, interleukin-33, which is released from liver sinusoidal endothelial cells, promotes NETosis of infiltrating neutrophils and exacerbates inflammatory injury [15]. Disruption of the miR-223 gene exacerbates acetaminophen-induced hepatic neutrophil infiltration, oxidative stress, and injury, and enhances TLR9 ligand-mediated activation of pro-inflammatory mediators in neutrophils [16]. However, it remains unknown about the mediator and underlying molecular mechanism of regulating neutrophil recruitment and activation during chronic liver injury.

The endocannabinoid system (ECS) comprises cannabinoid receptors (CBs; CB1 and CB2), endocannabinoids and their synthesis and degradation enzymes [17]. CB1 is highly expressed in central nervous system and is also found in the periphery, including immune system and liver at a lower level, while CB2 is mainly expressed in immune cells [18]. ECS has been proven to be involved in the regulation of multiple physiological processes, such as appetite control, energy balance, pain perception, and immune response [19–21]. Notably CBs have been identified as pivotal regulators of acute and chronic liver injury, especially in inflammation-related liver injury [22]. For examples, a potential impact of CB1 on the inflammatory response associated with NASH has been suggested by experiments in obese rats, showing that CB1 antagonist rimonabant plays a hepatoprotective role in the treatment of obesity-associated liver diseases and related features of metabolic syndrome [23,24]. Moreover, CB1 and CB2 participate in the resveratrol-induced anti-NASH effect by maintaining the gut barrier integrity and inhibiting gut inflammation in high-fat diet-induced NASH rat models [25]. Our previous studies have also found that CB1 promotes the infiltration and activation of bone marrow (BM)-derived monocytes/macrophages in carbon tetrachloride (CCl4)-induced liver injury mouse model, which could be inhibited by the blockade of CB1 [26,27]. However, the knowledge of whether CBs are involved in neutrophil function during sterile liver inflammation remains limited.

Here we investigate the effects of CBs on neutrophil chemotaxis and activation in isolated neutrophils and CCl4-induced murine models. Our findings suggest that CB1 but not CB2 mediates neutrophil chemotaxis and NETosis in vitro, which are dependent of ROS and MAPK signaling pathways. Furthermore, blockade of CB1 in vivo reduces the infiltration and activation of neutrophils and attenuates liver injury in CCl4-treated mice, which may represent an effective therapeutic strategy for liver diseases.

### **2. Materials and Methods**

#### *2.1. Materials*

RPMI Medium 1640 was from GIBCO/Invitrogen (Grand Island, NY, USA). PCR reagents were from Applied Biosystems. ACEA (special CB1 agonists), AM281 (CB1 antagonist), JWH133 (CB2 antagonist), SB203580 (p38 inhibitor) were from TOCRIS/R&D (Minneapolis, MN, USA). NAC and PTX were from Sigma-Aldrich (St. Louis, MO, USA). Fibronectin was from Calbiochem (Germany). YM254890 was from Adipogen Corp. (San Diego, CA, USA). SYTOX Green Nucleic Acid Stain was from Molecular Probes, Inc. (Eugene, OR, USA).

#### *2.2. Mouse Models of Liver Fibrosis*

A CCl4 (1 μL/g BW)/OO mixture (1:9 v/v) was injected into abdominal cavity of mice twice per week. Mice were sacrificed at 1, 2, and 3 days and 1, 2, and 4 weeks. The liver tissues were harvested. The intraperitoneal injection of AM281 (2.5 mg/kg BW) or DNAase I (11284932001, 50 μg/mouse, Roche, Swiss) was performed at 4 or 24 h before CCl4 administration. All animal work was conformed to the Ethics Committee of Capital Medical University and in accordance with the approved guidelines (approval number: AEEI-2014-131).

#### *2.3. BM Transplantation*

ICR male mice aged 6 weeks received lethal irradiation (8 Grays) and immediately received transplantation by a tail vein injection of 1.5 <sup>×</sup> 107 whole BM cells obtained from 3-week-old EGFP transgenic mice. Four weeks later, mice of BM-rebuild were subjected to CCl4-induced liver injury. After another 2 weeks, mice were sacrificed and liver tissues were harvested.

#### *2.4. FACS*

Non-parenchymal cells of mouse liver were isolated as described previously [28]. APC-Ly6G (BD Biosciences, Franklin Lakes, NJ, USA) and its isotype-matched negative control were added to the non-parenchymal cell suspension, respectively. After 15 min incubation in the dark, the cells were washed with PBS and subjected to FACS, which was performed on a FACSAria and analyzed with FACS Diva 4.1 (BD, Biosciences).

#### *2.5. Isolation of Mouse BM Neutrophils*

ICR mice aged 6 weeks were sacrificed by cervical dislocation at the time of neutrophils harvest. Tibias and femurs were removed and stripped of their muscles. The BM was flushed using PBS, and cell aggregates were disrupted via filtration through 70-μm cell strainer (BD Bioscience) and washed with PBS. Cell suspension was layered in a ratio of 1 to 3 on top of Histopaque 1077 (Sigma Aldrich), after centrifugation, precipitate was resuspended the with PBS. The cell suspension was layered in a ratio of 1 to 2 on top of Histopaque 1119 (Sigma Aldrich), after centrifugation, neutrophils were recovered on the top of Histopaque 1119. Neutrophils were washed with PBS and then resuspended in RPMI Medium 1640. The purity of neutrophils was determined by immunofluorescence staining for Ly6G (almost 100% cells were positive for Ly6G). Neutrophil viability was analyzed using Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) according to the manufacturer's procedure.

#### *2.6. Neutrophils Chemotaxis Assay*

Isolated bone marrow neutrophils were incubated with Calcein-AM (Life Technologies, CA, USA) to label cells and treated with AM281 (10 μM), NAC (5 mM), PTX (5 ng/mL), YM254890 (10 μM) or SB203580 (10 μM) for 20 min, then seeded to the upper chambers of a 3 μm-transwell (Corning). Then cells were allowed to migrate for another 2 h in the presence of ACEA (1 μM) or JWH133 (1 μM) in the lower chambers. The chambers were incubated at 37 ◦C in 5% CO2. Subsequently, chemotaxis of neutrophils was determined by the fluorescence value of Calcein-AM in the lower chambers, using a fluorescent plate reader EnVision 2104-0010 (Perkinelmer, MA, USA).

#### *2.7. Western Blot Analysis*

Proteins were extracted from cells (50 μg) or liver tissue (100 μg) using RIPA Lysis Buffer (R0010, Solarbio, China) added with Complete Protease Inhibitor Cocktail Tablets (04693116001, Roche, Swiss). The components of RIPA Lysis Buffer were as follows: 50 mM Tris (pH 7.4), 150 mM NaCl, 1%TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM sodium pyrophosphate, 25 mM b-glycerophosphate, 1 mM EDTA, 1mM Na3VO4, 0.5 mg/mL leupeptin. Then the extract was separated by SDS-PAGE and subjected to Western blot analysis. Membranes were incubated overnight using the following antibodies: rabbit anti-citrullinated-histone H3 (CitH3) polyclonal antibody (ab5103, 1:200, Abcam, Cambridge, United Kingdom), rabbit anti-ERK1/2 monoclonal antibody (4695) and rabbit anti-phorspho-ERK1/2 monoclonal antibody (4376, 1:1000, Cell Signaling, Beverly, MA, USA); rabbit anti-p38 polyclonal antibody (9212) and rabbit anti-phorspho-p38 polyclonal antibody (9211, 1:1000, Cell Signaling); rabbit anti-JNK polyclonal antibody (9252) and rabbit anti-phospho-JNK monoclonal antibody (4668, 1:1000, Cell Signaling); mouse anti-β-tubulin (HC101, 1:1000, TransGen Biotech, China) and anti-β-actin monoclonal antibodies (HC201, 1:1000, TransGen Biotech, China). IRDye 800CW Goat anti-Mouse IgG (H + L) Secondary Antibody (92632210), IRDye 800CW Goat anti-Rabbit IgG (H + L) Secondary Antibody (92632211, 1:10000, LI-COR, NE, USA) were used. The bands were displayed using ODYSSEY and quantified by Odyssey v3.0 software. β-tubulin or β-actin were as references.
