IRG1 Promotes Inflammation Resolution By Enhancing Neutrophil Reverse Migration

Background and objective:Neutrophils play a central role in the development and resolution of inflammation.Thus,neutrophils were regarded as the target for therapeutic interventions.Neutrophil reverse migration is a recently identified phenomenon in which neutrophil migrate away from the inflammatory site back into the vasculature following the initial neutrophil infiltration.Reverse migrated neutrophils showed significant different phenotype from the identified neutrophils in circulation or inflammatory site.Due to the limited number of these cells,the mechanism and effect of neutrophil reverse migration were still unclear.By single cell RNA sequencing on the leukocytes in inflammatory site and circulation,present study was aimed to explore the neutrophil heterogeneity during the inflammatory responses,and further investigate the mechanism in neutrophil reverse migration.Method and results:Part 1:Single cell RNA sequencing reveals the heterogeneity of neutrophils Bronchoalveolar lavage fluid leukocytes and circulation leukocytes from the mice with LPS intratracheal treatment after 0 h,6 h and 24 h were collected for single cell RNA sequencing.Reversed Graph Embedding analysis showed that there were two fates of neutrophils:one was developed to amplify the inflammatory response,while another was developed to enhance the phagocytosis and inhibit inflammatory process.Part 2:Characterization of reverse-migrated PMNs(rM-ed PMNs)Compared with the other neutrophils in the blood,reverse migrated neutrophils showed higher expression of C-C motif chemokine receptor like 2(CCRL2),one non-canonical chemokine receptor.We further developed reverse migrated cells tracking system on mice air pouch model.Compared with the circulation neutrophils,reverse migrated neutrophils showed increased membrane expression of CCRL2.Meanwhile,chemerin,the ligand of CCRL2,was increased in the mice plasma.The ratio of reverse migrated neutrophil was decreased by treating the mice with chemerin neutralization antibody by tail vein injection,suggesting that chemerin could induce the neutrophil reverse migration by interacting with CCRL2.Part 3:IRG1 promotes inflammation resolution by enhancing neutrophil reverse migration.To further investigate the mechanism of CCRL2,we analyzed the transcripts expression correlation between CCRL2 and other genes from single cell RNA sequencing.Immune response gene(IRG1)transcripts was highly positive related with the transcripts of CCRL2.Compared with wild type mice,IRG1 knock out mice showed decreased reverse migrated neutrophil ratio and increased inflammatory mediator concentration.Furthermore,IRG1 was found to decrease the Intercellular adhesion molecule 1(ICAM1)expression on neutrophil membrane.ICAM1 neutralization antibody could increase the reverse migrated neutrophil ratio,suggesting that IRG1 could promote neutrophil reverse migration and inflammation resolution by decreasing ICAM1 expression on neutrophil membrane.Conclusion:In the late stage of inflammatory process,chemerin concentration was increased in mice plasma.By interacting with CCRL2 on neutrophils,chemerin could induce the neutrophil leave the inflammatory loci.IRG1 could decrease the ICAM1 membrane expression,which promoted neutrophil reverse migration and inflammation resolution.