*3.1. Numerous Neutrophils Are Recruited and Activated in the Liver of CCl4-Treated Mice*

To investigate the dynamic change of neutrophil signatures in sterile liver inflammation, we examined the mRNA expression of neutrophil marker Ly6G in the liver treated by CCl4 for different time points. Our results showed that Ly6G mRNA expression up-regulated from 7 days of CCl4 administration and reached the peak at 2 weeks, whereas the expression evidently decreased at 4 weeks compared with 2 weeks (Figure 1A), indicating that numerous neutrophils were recruited to injured liver during the early stage of chronic liver injury. Further, FACS analysis revealed that percentage of Ly6G<sup>+</sup> neutrophils was much higher in CCl4-treated mice for 2 weeks compared with that in olive oil (OO)-treated mice (Figure 1B,C).

To clarify the origin of neutrophils recruited to the injured liver, we performed a genetic EGFP-labeled BM cell transplantation to the mice that had been lethally irradiated. Then the chimeric mice received intraperitoneal injection of CCl4 for 2 weeks to induce liver injury. We isolated hepatic non-parenchymal cells from liver tissue and detected Ly6G<sup>+</sup> cells by FACS. The percentage of Ly6G+EGFP<sup>+</sup> neutrophils (BM origin, OO group: 1.81%; CCl4 group: 12.00%) was much higher than Ly6G+EGFP<sup>−</sup> neutrophils (non-BM origin, OO group: 0.07%; CCl4 group: 0.13%) in both OO and CCl4 groups (Figure 1D,E). Moreover, Ly6G+EGFP<sup>+</sup> neutrophils were significantly increased after CCl4 administration compared with that in OO group (Figure 1D,E), indicating the recruited neutrophils in injured liver were mostly derived from BM. Then we performed immunofluorescent staining to examine CitH3 expression in the neutrophils of injured liver (Figure 1F). Further, increased hepatic level of citrullinated histone H3 (CitH3, specific marker of NETosis) was detected in CCl4-treated mice (Figure 1G,H), suggesting the activation of these infiltrating neutrophils in the injured liver. Correlation analysis showed a positive correlation between CitH3 protein levels and Ly6G mRNA expression in liver tissue (Figure 1I). Altogether these results demonstrate that large numbers of BM-derived neutrophils are recruited and activated in the early stage of chronic liver injury.

#### *3.2. CB Expression Positively Correlates with Neutrophil Signatures in CCl4-Treated Mice, and CBs Are Abundantly Expressed in Isolated Neutrophils*

Our previous study had showed that CB1 and CB2 expression were increased in CCl4-induced liver injury [27]. Here we undertook correlation analysis of mRNA expression levels between CB1 or CB2 and Ly6G. Each dot represented one liver sample from all mice (including OO and CCl4-treated groups). Correlation coefficients were calculated using relative mRNA expression levels of CB1/CB2 and Ly6G from the same sample by Pearson correlation test (Figure 2A). Although both CB1 and CB2 were positively correlated with Ly6G (*p* < 0.05), CB1 represented a particularly higher correlation coefficient (Figure 2A). Based on the prevailing amount of BM-derived neutrophils in injured liver, we used mouse neutrophils isolated from BM in subsequent cellular experiments. The expression of CB1 and CB2 in mouse BM derived-neutrophils were detected at mRNA level (Figure 2B), and protein

level by immunofluorscence (Figure 2C). These results demonstrated the positive correlation between the expression of CB1 and neutrophil signatures in CCl4-treated mice and the abundant expression of CB1 in isolated neutrophils, suggesting that CB1 might play an important role in the recruitment and activation of neutrophils during sterile liver injury.

**Figure 1.** Numerous neutrophils are recruited and activated in the liver of carbon tetrachloride (CCl4)-treated mice. (**A**) The mRNA expression of neutrophil marker Ly6G was examined by qRT-PCR in the injured liver of CCl4 mice. (**B**,**C**) Representative FACS plots and quantification for total neutrophils (Ly6G+). (**D**,**E**) Representative FACS plots and quantification for neutrophils of BM origin (Ly6G+EGFP+) and non-BM origin (Ly6G+EGFP−). (**F**) Immunofluorescent staining for CitH3 in the liver of CCl4-treated mice. Scale bars, 20 μm. (**G**,**H**) CitH3 expression in the injured liver was examined by Western blot. (**I**) The correlation between CitH3 protein levels and Ly6G mRNA expression in liver tissue. Data are presented as the mean ± SEM. N = 6 per group. \* *p* < 0.05 vs. control. # *p* < 0.05 vs. EGFP<sup>−</sup> neutrophils with the same treatment.

**Figure 2.** Cannabinoid receptor (CB) expression positively correlates with neutrophil signatures in CCl4-treated mice, and CBs are abundantly expressed in isolated neutrophils. (**A**) The correlation between Ly6G and CB1 or CB2 in liver tissue. (**B**) The amplification plots of CB1 and CB2 expression in neutrophils by RT-qPCR. (**C**) Representative images of immunofluorescent staining for Ly6G (green) and CB1 or CB2 (red) in neutrophils. The nuclei were stained with DAPI (blue). Scale bars, 20 μm. N = 6 per group.

### *3.3. CB1 Rather than CB2 Mediates the Chemotaxis and Cytoskeletal Remodeling of Neutrophils In Vitro*

Transwell assay was performed to explore whether CBs were involved in the chemotaxis of neutrophils in vitro. Treatment with ACEA (CB1 agonist) significantly increased the migration capacity of neutrophils in a dose-dependent manner, while JWH133 (CB2 agonist) had no such effect (Figure 3A). Due to the weak affinity between ACEA and CB2, we pretreated neutrophils with CB1 antagonist AM281 (1 and 10 μM) in ACEA-stimulated cells and showed declined chemotaxis of neutrophils with AM281 pretreatment (Figure 3B), further proving that CB1 mediated the chemotaxis of neutrophils.

**Figure 3.** CB1 rather than CB2 mediates the chemotaxis and cytoskeletal remodeling of neutrophils in vitro. Chemotaxis assays were performed by transwell chambers. (**A**) Neutrophil chemotaxis with ACEA (CB1 agonist) or JWH133 (CB2 agonist) treatment for 2 h. (**B**) Effect of AM281 (CB1 antagonist) on neutrophil chemotaxis. (**C**) Representative images of F-actin remodeling with ACEA (1 μM, 2 h) and JWH 133 (1 μM, 2 h) treatment in neutrophils. Scale bars, 20 μm. (**D**) Quantification of F-actin with or without AM281 (10 μM) in ACEA-treated neutrophils. (**E**) The total fiber area was qualified by high content analysis in ACEA-treated neutrophils with or without AM281 pretreatment. Data are presented as the mean ± SEM. N = 5 per group. \* *p* < 0.05 vs. control. # *p* < 0.05 vs. ACEA-treated alone.

Cytoskeletal remodeling is a prerequisite for cell chemotaxis and migration [29]. To evaluate the involvement of CB1 in cytoskeletal remodeling, neutrophils were stimulated with ACEA or JWH133 and then stained with FITC-conjugated phalloidin. Our findings indicated that ACEA-treated neutrophils were able to form a well-defined F-actin-rich leading edge (Figure 3C) and induced an increase in F-actin content (Figure 3D), whereas JWH133-treated neutrophils did not show obvious polymerized F-actin (Figure 3C,D). Consistent with the chemotaxis results above, AM281 could significantly reduce the increase of F-actin content induced by ACEA (Figure 3D). Moreover, the amount and distribution of actin fibers in neutrophils were determined by high content analysis. Treatment with ACEA showed significant increases in the total fiber area, which can be reversed by AM281 (Figure 3E). These results support that CB1 rather than CB2 plays an important role in neutrophil chemotaxis and cytoskeletal remodeling.

#### *3.4. CB1 but not CB2 is Involved in the Activation of Neutrophils In Vitro*

Next we sought to determine whether CBs played a role in the activation of neutrophils, including NETosis, MPO release from lysosome and ROS burst. Neutrophils stimulated with ACEA exhibited increased CitH3 fluorescence compared with untreated cells and represented manifest web-like chromatin release, in which chromatin and CitH3 (red) had good co-localization in neutrophils (Ly6G+, violet) (Figure 4A,B), and can be suppressed by AM281 (Figure 4B). Western blot results showed the same effect of ACEA on CitH3 expression (Figure 4C). In contrast, JWH133-treated neutrophils did not exhibit increased level of CitH3 or web-like chromatin release (Figure 4A–C). We performed CCK-8 assay to measure neutrophil viability under the treatment of ACEA, the condition to induce NETosis. Our results showed that ACEA decreased the cell viability of neutrophils, while CB2 agonist JWH133 had no such effect, which was in accordance with the results of NETosis (Figure 4D).

**Figure 4.** CB1 but not CB2 is involved in CitH3 expression and NETosis in vitro. (**A**) Representative confocal images of CitH3 (red) and Ly6G (violet) immunofluorescent staining and NETosis in ACEA or JWH133-treated neutrophils. The nuclei were stained with SYTOX® Green (green) and DAPI (blue). Scale bars, 20 μm. (**B**) Quantification of CitH3 and NETosis in ACEA or JWH133-treated neutrophils. (**C**) CitH3 protein level in ACEA or JWH133-stimulated neutrophils was examined by Western blot. (**D**) Cell viability of neutrophils treated by ACEA or JWH-133 by CCK-8 assay. Data are presented as the mean ± SEM. N = 4 per group. \* *p* < 0.05 vs. control. # *p* < 0.05 vs. ACEA-treated alone.

Normally, MPO exists in lysosome and is undetectable by antibodies, when stimulated MPO is released from lysosome becoming detectable [30]. Further we detected the release of MPO in neutrophils by immunofluorescence. ACEA treatment resulted in more MPO release of neutrophils compared with control, while JWH133 treatment had no such effect (Figure 5A,B). Similar to the results of CitH3, AM281 also blocked ACEA-induced MPO release in neutrophils (Figure 5B). We then measured ROS burst in neutrophils with ACEA treatment. In response to ACEA stimulation, neutrophils showed a significant increase of ROS burst at 10 min, and this increase could be significantly prevented by pre-incubation with NAC which is the scavenger of ROS (Figure 5C). In addition, pretreatment with AM281 repressed ACEA-induced ROS burst, and JWH133 could not induce ROS burst in neutrophils (Figure 5D). ROS immunofluorescence images also displayed the increase of ROS burst by ACEA treatment (Figure 5E). Altogether these results display that CB1 but not CB2 mediates the activation of neutrophils.

**Figure 5.** CB1 but not CB2 mediates MPO release and ROS burst in vitro. (**A**) Representative confocal images myeloperoxidase (MPO) (red) and Ly6G (violet) immunofluorescent staining in ACEA or JWH133-stimulated neutrophils. The nuclei were stained with SYTOX® Green (green) and DAPI (blue). Scale bars, 20μm. (**B**) Quantification of MPO in ACEA or JWH133-treated neutrophils. (**C**) ROS burst in ACEA-treated neutrophils with or without NAC. (**D**) ROS burst in ACEA-treated neutrophils with or without AM281. (**E**) ROS immunofluorescence images in ACEA- or JWH-133-treated neutrophils. Data are presented as the mean ± SEM. N = 4 per group. \* *p* < 0.05 vs. control. # *p* < 0.05 vs. ACEA-treated alone.
