Effect of autophagy on expression of neutrophil programmed death ligand-1 in mice with sepsis

Abstract

To investigate the effect and mechanism of autophagy on the expression of neutrophil programmed death ligand-1 (PD-L1) in mice with sepsis. (1) In vivo experiment: male C57BL/6 mice aged 6-8 weeks were divided into sham operation group (Sham group), cecum ligation and perforation (CLP) group, and rapamycin (RAP)+CLP group by random number table with 10 mice in each group. The sepsis model was reproduced by CLP, and the cecum and perforation were not ligated in Sham group, and other operations were the same as CLP group. The mice in RAP+CLP group were intraperitoneally injected with autophagy agonist RAP 4 mg×kg-1×d-1 7 days before modeling, while the mice in Sham group and CLP group were not treated. Lung, liver, spleen and pancreas tissues were harvested for immunohistochemical staining 4 days after the operation, and the infiltration of neutrophils in various organs was observed under light microscope. Meanwhile, the expressions of immunosuppressive molecule PD-L1 and autophagy marker microtubule-associated protein 1 light chain 3 (LC3) in lung neutrophils were determined by immunofluorescence staining. (2) In vitro experiment: mouse bone marrow neutrophils were extracted and re-suspended to 1×1010/L, and they were divided into blank control group (without any treatment), RAP control group (RAP 100 μmol/L), autophagy inhibitor Bafilomycin A1 (Baf) control group (Baf 10 μmol/L), lipopolysaccharide (LPS) stimulation group (LPS 1 mg /L), RAP+LPS group, and Baf+LPS group. The latter two groups were pretreated with 100 μmol/L RAP or 10 μmol/L Baf 30 minutes before LPS stimulation, respectively. The expression of PD-L1 mRNA of neutrophils was determined by reverse transcription-polymerase chain reaction (RT-PCR) at 0, 4, 12 hours after LPS stimulation. At the same time, the expressions of PD-L1, LC3 and p62 at the protein level were determined by Western Blot. (1) In vivo experiment: according to immunohistochemical experiments, a large amount of infiltration of neutrophils in lung, liver, spleen and pancreas was found at 4 hours after CLP. In the immunofluorescence, with the time extension after CLP, the positive expression of LC3 in the lung tissue showed a decreased tendency, and PD-L1 expression was significantly increased. RAP pretreatment could promote the expression of LC3 and reduce the expression of PD-L1 in CLP mice. (2) In vitro experiment: in terms of mRNA levels, with the extension of LPS stimulation time, the expression of PD-L1 mRNA in mouse neutrophils was increased continuously, and peaked at 12 hours, it was significantly higher than that in the blank control group (2-ΔΔCT: 72.2±10.0 vs. 13.0±0.8, P < 0.01). Compared with LPS stimulation group, the expression of PD-L1 mRNA in RAP+LPS group was significantly down-regulated [12-hour PD-L1 mRNA (2-ΔΔCT): 47.4±7.3 vs. 72.2±10.0, P < 0.01]. In Baf+LPS group, PD-L1 mRNA expression was significantly up-regulated as compared with that in LPS stimulation group [12-hour PD-L1 mRNA (2-ΔΔCT): 109.1±7.4 vs. 72.2±10.0, P < 0.01]. At the protein levels, at 4 hours after LPS stimulation, the positive expressions of PD-L1, LC3 and p62 were increased significantly as compared with those in the blank control group, and PD-L1 and p62 were increased continuously with time. Compared with the LPS stimulation group, the expressions of PD-L1 and p62 in the RAP+LPS group were significantly down-regulated, while the expression of LC3 was continually increased, indicating that the level of autophagy was increased, and autophagy was circulated smoothly. On the contrary, the expressions of PD-L1, LC3 and p62 in the Baf+LPS group were significantly up-regulated, indicating that the binding of autophagy and lysosome was blocked, and autophagy was not smooth. In sepsis, the infiltration of neutrophils in all organs increased, and the expression of PD-L1 of neutrophils in lungs was increased significantly, while the expression level of autophagy was decreased. The expression of PD-L1 stimulated by LPS can be inhibited by autophagy agonists, and promoted by autophagy inhibitors. PD-L1 has a negative regulatory effect on sepsis. It can reduce the expression of PD-L1 molecule in sepsis by targeting autophagy, so as to improve sepsis.