Mechanism Of Autophagy Regulating PD-L1 Of Neutrophils In Sepsis Mice

Objective To investigate the regulation of programmed death ligand-1(PD-L1)in mice by changes in autophagy of neutrophils during sepsis.Methods The weighted gene co-expression analysis method was used to analyze the differential genes detected by the Affymetrix Mouse Transcriptome Array 1.0 gene chip.The co-expression modules were constructed based on the dissimilarity coefficients.The GO term and KEGG pathway enrichment analysis were performed on the co-expression moduLes.Look for phenotypes that are closely related to changes in neutrophil function during sepsis.The cecal ligation and perforation(CLP)experiment was used in vivo to simulate the sepsis mouse model.Four organs(lung,liver,spleen,pancreas)were removed for immunohistochemical staining 4 days after CLP,and the infiltration of neutrophils in each organ was observed.Meanwhile,immunofluorescence staining was used to detect the expression levels of neutrophils PD-L1 and autophagy marker LC3 in lungs of CLP mice at 4 days.With the intervention of autophagy agonist Rapamycin(RAPA),the expression levels of PD-L1 and autophagy marker LC3 in the lung neutrophils of CLP mice were measured at 1 and 4 days after surgery.Lipopolysaccharide(LPS)was used to stimulate mouse bone marrow neutrophils to simulate the sepsis model,and autophagy agonist Rapamycin and autophagy inhibitor Bafilomycin A1(Baf)were used for intervention stimulation.PD-L1 and LC3 expression levels were detected at the gene and protein levels by PCR and Western Blot respectively.Meanwhile,flow cytometry and CCK8 experiment were used to detect the apoptosis of neutrophils.ResuLts The differential genes were divided into 9 modules with different functions,and two moduLes closely related to sepsis were found,namely the blue and pink modules.The GO term and KEGG pathway enrichment analysis were performed on the blue and pink modules.It was found that autophagy is closely related to changes in neutrophilfunction during sepsis.At the same time,the apoptotic function of neutrophils was partially restored in sepsis after autophagy was promoted by the use of autophagy agonist RAPA.In vitro experiments found that PD-L1 expression increased and autophagy decreased during sepsis.When LPS and RAPA were used simultaneously,the expression level of PD-L1 molecule was significantly reduced.In contrast,when LPS and Baf were used simultaneously,the expression level of PD-L1 molecules increased significantly.In vivo experiments found that neutrophil infiltration in important organs increased during sepsis.Immunofluorescence showed that PD-L1 expression in lung neutrophils of CLP mice increased,and the expression of autophagy marker LC3 decreased.After administration of mouse RAPA,lung neutrophil PD-L1 expression decreased,LC3 expression increased.Conclusions Through WGCNA analysis,we found that autophagy is closely related to the changes in neutrophil function during sepsis.In vivo and in vitro studies,we have found that during sepsis,infiltration of neutrophils in important organs increases,and the expression of PD-L1 is significantly up-regulated.After stimulating with autophagy agonist RAPA,the expression of PD-L1 in neutrophils can be significantly down-regulated.PD-L1 is a well-known immune checkpoint molecule,and its up-regulation has a negative regulatory effect on sepsis.Targeting autophagy can significantly reduce the expression of PD-L1 molecules on the surface of neutrophils.At the same time,the apoptosis of neutrophils was increased and the sepsis was improved.