*3.6. G*α*i*/*<sup>o</sup> Signal Is Involved in CB1-Mediated Chemotaxis and NETosis In Vitro*

CB1 is a G-protein-coupled receptor which can transduce corresponding G protein-related signaling. To determine the distinct G-protein subtype involved in neutrophil chemotaxis and NETosis, we pre-treated cells with pertussis toxin (PTX) (Gαi/<sup>o</sup> inhibitor) or YM254890 (Gα<sup>q</sup> inhibitor). PTX prevented ACEA-induced neutrophil chemotaxis, while YM254890 exhibited no effect on it (Figure 7A). In line with this, the up-regulation of Cit-H3 protein induced by ACEA can be reversed by PTX, not YM254890 (Figure 7B–D). Moreover, less MPO release of neutrophils was observed after the pre-incubation of PTX in ACEA-stimulated cells compared with control, while YM254890 treatment had no such effect (Figure 7E). Markedly, PTX pretreatment inhibited ACEA-induced ROS burst, while YM254890 could not reduce ROS burst in neutrophils (Figure 7F). Taken together, these results indicate that Gαi/o, not Gα<sup>q</sup> signal is involved in CB1-mediated chemotaxis and NETosis.

**Figure 7.** Gαi/<sup>o</sup> signal is involved in CB1-mediated chemotaxis and NETosis in vitro. (**A**) ACEA-induced neutrophil chemotaxis pretreatedwith Gαi/<sup>o</sup> inhibitor PTX or Gα<sup>q</sup> inhibitor YM254890. (**B**,**C**) Representative images and quantification of CitH3 immunofluorescent staining (green) and NETosis in ACEA-treated neutrophils pretreated with PTX or YM254890. The nuclei were stained with DAPI (blue). Scale bars, 20 μm. (**D**) CitH3 protein level with PTX or YM254890 pretreatment was examined by Western blot. (**E**) Quantification of myeloperoxidase (MPO) immunofluorescence with pertussis toxin (PTX) or YM254890 pretreatment. (**F**) ROS burst in ACEA-treated neutrophils with or without PTX or YM254890 pretreatment. Data are presented as the mean ± SEM. N = 4 per group. \* *p* < 0.05 vs. control. # *p* < 0.05 vs. ACEA-treated alone.

#### *3.7. ROS Is Required for CB1-Mediated Neutrophil Chemotaxis and NETosis In Vitro*

Since ROS burst was markedly induced by ACEA, we then explored whether ROS was involved in CB1-mediated neutrophil chemotaxis and NETosis. Transwell assay showed that CB1-mediated chemotaxis in neutrophils was suppressed by NAC (Figure 8A), suggesting that ROS acted as a signaling molecule in CB1-mediated neutrophil chemotaxis. As NETosis can either be ROS-dependent or ROS-independent [31,32], we pre-treated neutrophils with NAC before stimulation with ACEA and detected CitH3 expression and MPO release, to investigate whether CB1-induced NETosis required ROS in vitro. NAC significantly blocked ACEA-induced increase of CitH3 detected by immunofluorescence (Figure 8B,C) and Western blot (Figure 8D). Similarly, lower MPO fluorescence was observed in neutrophils pre-incubated with NAC before ACEA stimulation compared with that without NAC (Figure 8E). Collectively, elimination of ROS suppresses CB1-mediated chemotaxis and NETosis in neutrophils, suggesting that ROS acts as an important signaling molecule in CB1-mediated neutrophil function.

**Figure 8.** ROS is required in CB1-mediated chemotaxis and NETosis in vitro. (**A**) ACEA-induced neutrophil chemotaxis pretreated with or without NAC (5 mM). (**B**,**C**) Representative images and quantification of CitH3 immunofluorescent staining (green) and NETosis in ACEA-treated neutrophils pretreated with or without NAC. The nuclei were stained with DAPI (blue). Scale bars, 20 μm. (**D**) CitH3 protein level with or without NAC pretreatment was examined by Western blot. (**E**) Quantification of MPO immunofluorescence with or without NAC pretreatment. Data are presented as the mean ± SEM. N = 4 per group. \* *p* < 0.05 vs. control. # *p* < 0.05 vs. ACEA-treated alone.

#### *3.8. p38 MAPK Signaling Pathway, Located In the Downstream of ROS, Is Involved in CB1-Mediated Neutrophil Chemotaxis and NETosis*

CB1 is a G-protein-coupled receptor whose biological function depends on multiple signaling pathways, such as AMPK and MAPK signaling pathways [33]. To identify which pathway controls the chemotaxis and NETosis of neutrophils, we first detected the phosphorylation of p38, JNK and ERK after ACEA treatment. Stimulation with ACEA led to a significant increase in the protein level of phosphor-p38 (Figure 9A,B), but failed to activate JNK (Figure 9C,D) and ERK (Figure 9E,F) in neutrophils. Based on these, we focused on the key role of p38 in neutrophil function in the following experiments.

**Figure 9.** ACEA increases the protein level of phosphor-p38 in neutrophils. (**A**,**B**) Phosphor-p38 and total p38 expression after ACEA treatment was measured by Western blot. (**C**,**D**) Phosphor-JNK and total JNK expression. (**E**,**F**) Phosphor-ERK and total ERK expression.Data are presented as the mean ± SEM. N = 3 per group. \* *p* < 0.05 vs. control.

Moreover, ACEA-induced neutrophil chemotaxis was significantly suppressed by p38 inhibitor SB203580 (Figure 10A), indicating the key role of p38 in neutrophil chemotaxis. In case of CB1-mediated NETosis, p38 inhibition restrained CitH3 expression detected by both fluorescence (Figure 10B,C) and Western blot (Figure 10D). Similarly, lower MPO fluorescence was observed in neutrophils pre-incubated with p38 inhibitor before ACEA stimulation (Figure 10E). These results indicate that CB1-mediated p38 phosphorylation is involved in neutrophil chemotaxis and NETosis. Furthermore, inhibition of p38 had no effect on the production of ROS (Figure 10F), whereas elimination of ROS inhibited CB1-mediated phosphorylation of p38 (Figure 10G), suggesting that ROS was upstream

of p38. Combining the above results, p38 MAPK signaling pathway is required for CB1-mediated neutrophil chemotaxis and NETosis, and locates in the downstream of ROS (Figure 10H).

**Figure 10.** p38 MAPK signaling pathway is involved in CB1-mediated neutrophil chemotaxis and NETosis, and located in the downstream of ROS. (**A**) ACEA-induced neutrophil chemotaxis with or without p38 inhibitor SB203580 (10 μM). (**B**,**C**) Quantification of CitH3 immunofluorescence with or without SB203580. Scale bars, 20 μm. (**D**) CitH3 protein level with or without SB203580 was examined by Western blot. (**E**) Quantification of MPO immunofluorescence with or without SB203580. (**F**) ROS burst in ACEA-treated neutrophils with or without SB203580. (**G**) Phosphor-p38 and total p38 expression was measured by Western blot with or without NAC. (**H**) Schema graph of CB1 in neutrophil chemotaxis and NETosis. Data are presented as the mean ± SEM. N = 4 per group. \* *p* < 0.05 vs. control. # *p* < 0.05 vs. ACEA-treated alone.

#### **4. Discussion**

In summary, this study demonstrates for the first time that CB1 mediates neutrophil chemotaxis and activation in a ROS- and p38 MAPK-dependent manner in sterile liver inflammation. Our work provides several new findings as follows: 1. Numerous bone marrow-derived neutrophils are recruited and activated in the liver of CCl4-treated mice; 2. CBs positively correlate with neutrophil signatures in CCl4-treated mice, and are abundantly expressed in isolated neutrophils; 3. In vitro CB1 rather than CB2 mediates neutrophil chemotaxis, NETosis, MPO release and ROS burst via Gαi/<sup>o</sup> signal; 4. ROS and p38 MAPK signaling pathway are both required for CB1-mediated neutrophil chemotaxis and NETosis, and p38 MAPK signaling pathway locates in the downstream of ROS; 5. Blockade of CB1 significantly attenuates neutrophil infiltration and liver inflammation in CCl4-treated mice.

Neutrophils act as the first responders of the innate immune system and their crucial role in fighting invading pathogens during bacterial inflammation has been well established by published literature [34,35]. Recently more and more studies have been focusing on the prevailing role of neutrophils in sterile inflammation, and overexuberant neutrophil recruitment is associated with collateral tissue damage, defective healing, and chronic inflammation [36,37]. In the current study, we show that numerous BM-derived neutrophils are recruited to the site of liver injury shortly. The hepatic levels of neutrophil marker Ly6G begin to rise from 7 days of CCl4 administration and peek at 2 weeks, which is the early stage of chronic liver injury. This is in agreement with published studies demonstrating that neutrophil depletion by injection of Ly6G antibody markedly reduces chronic-binge ethanol feeding-induced liver injury and liver transplantation ischemia–reperfusion injury [8,38,39]. However, there are studies identifying the dual role for neutrophils in acetaminophen-induced acute liver injury, with neutrophil-mediated injury amplification early on, but exerting protective effects during the repair phase as depletion of neutrophils increases liver damage [40,41]. Also neutrophils contribute to spontaneous resolution of liver inflammation and fibrosis via microRNA-223 in CCl4-induced chronic liver injury [41]. More studies will be needed to figure out the mechanism underlying differential role of neutrophils in different models of liver inflammation and fibrosis.

ECS is implicated in the pathogenesis of numerous diseases, including cancer, cardiovascular disease, and liver disease [42,43]. Especially CB1 has emerged as a pivotal mediator in liver and exerts profibrogenic effects in chronic liver diseases including hepatic fibrosis, liver cirrhosis alcoholic fatty liver and nonalcoholic fatty liver [44]. Our previous studies have also demonstrated the vital role of CB1 in the migration and activation of BM-derived mesenchymal stromal cells and monocytes/macrophages in CCl4-induced chronic liver injury [26,27,45,46]. However, the effect of CBs on neutrophil function during sterile liver inflammation is unclear up to now and is first documented in the present study. Our data display that CB1 rather than CB2 mediates the chemotaxis of neutrophils and NETosis, and CB1 blockade with AM281 reduces the infiltration and NETosis of neutrophils and attenuates liver injury in vivo, which can be used as a novel target for the treatment of liver fibrosis.

NETs, DNA webs released into the extracellular environment by activated neutrophils, are thought to play a key role in the function of neutrophils [47,48]. Unlike nuclear chromatin, NETs are highly decondensed chromatin structures, and PAD4 has been reported to be essential in chromatin decondensation to form NETs by catalyzing histone citrullination [49]. The partial PAD4-deficiency (Pad4+/–) reduced acute lung injury induced by bacteria and improved survival, while complete NET inhibition by PAD4 deficiency (Pad4–/–) reduced lung injury [50]. Further studies will be needed to investigate PAD4 expression and the effect of PAD4 on NETosis in our CCl4-treated mice and ACEA-treated neutrophils.

NETosis was initially found dependent on the ROS by NADPH [32], and was subsequently found also to be independent of ROS [31]. In the present study, ACEA-mediated neutrophil chemotaxis and NETosis can be significantly suppressed by ROS scavenger NAC, indicating that CB1 induces the chemotaxis and NETosis of neutrophils in a ROS-dependent manner. Since ROS could activate MAPK pathway and then mediate PMA-induced NETosis [51], here we detect the activation of p38, JNK, and ERK after ACEA stimulation, showing that only p38 MAPK pathway is activated and involved in

CB1-mediated neutrophil chemotaxis and NETosis. Further we identify the upstream and downstream relationship of ROS and p38 MAPK signaling pathways by the fact that p38 inhibition has no effect on the production of ROS, whereas ROS elimination inhibits CB1-mediated phosphorylation of p38, suggesting that ROS acts as an upstream signaling molecule of p38 MAPK in neutrophil chemotaxis and NETosis.

In conclusion, we identify the critical role of CB1 in neutrophil chemotaxis and NETosis during sterile liver inflammation and explore the underlying mechanism associated with Gαi/o/ROS/p38 MAPK signaling pathway, which may open new perspectives for pharmacological treatment of liver disease.

**Author Contributions:** Conceptualization, L.L. (Lin Yang); Methodology, X.Z. (Xuan Zhou), X.F. and X.Z. (Xinhao Zhao); Formal Analysis, X.Z. (Xuan Zhou), L.Y. (Le Yang) and N.C.; Investigation, X.Z. (Xuan Zhou), X.F. and X.Z. (Xinhao Zhao); Writing—Original Draft Preparation, X.Z. (Xuan Zhou) and L.Y. (Le Yang); Writing—Review and Editing, L.L. (Lin Yang); Project Administration, L.Y. (Lin Yang); Funding Acquisition, L.L. (Lin Yang). All authors have read and agree to the published version of the manuscript.

**Funding:** This research was funded by National Natural and Science Foundation of China (81670550, 81430013), Beijing Natural Science Foundation (7172019).

**Conflicts of Interest:** The authors declare no conflict of interest.
