**Xuan Zhou** †**, Le Yang** †**, Xiaoting Fan** †**, Xinhao Zhao, Na Chang, Lin Yang and Liying Li \***

Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing 100069, China; zhouxuanyee@126.com (X.Z.); yangle@ccmu.edu.cn (L.Y.); 18210564428@163.com (X.F.); xinhaozhao0010@163.com (X.Z.); changna@ccmu.edu.cn (N.C.); yang\_lin@ccmu.edu.cn (L.Y.)

**\*** Correspondence: liliying@ccmu.edu.cn

† These authors contributed equally.

Received: 22 January 2020; Accepted: 3 February 2020; Published: 5 February 2020

**Abstract:** Neutrophils play an essential role in the control of inflammatory diseases. However, whether cannabinoid receptors (CBs) play a role in neutrophil chemotaxis and NETosis in sterile liver inflammation remains unknown. The expression of marker genes on neutrophils was characterized by FACS, immunofluorescence, qRT-PCR, and Western blot. The amount of neutrophils was significantly elevated from 7 days and reached the peak at 2 weeks in carbon tetrachloride (CCl4)-treated mouse liver. The mRNA expression of neutrophil marker Ly6G had positive correlation with CB1 and CB2 expression in injured liver. In vitro CBs were abundantly expressed in isolated neutrophils and CB1 agonist ACEA promoted the chemotaxis and cytoskeletal remodeling, which can be suppressed by CB1 antagonist AM281. Moreover, ACEA induced NETosis, myeloperoxidase release from lysosome and ROS burst, indicating neutrophil activation, via Gαi/o. Conversely, CB2 agonist JWH133 had no effect on neutrophil function. ROS and p38 MAPK signaling pathways were involved in CB1-mediated neutrophil function, and ROS was upstream of p38 MAPK. CB1 blockade in vivo significantly attenuated neutrophil infiltration and liver inflammation in CCl4-treated mice. Taken together, CB1 mediates neutrophil chemotaxis and NETosis via Gαi/o/ROS/p38 MAPK signaling pathway in liver inflammation, which represents an effective therapeutic strategy for liver diseases.

**Keywords:** liver injury; neutrophil extracellular trap; myeloperoxidase; carbon tetrachloride
