Platelet-derived Serotonin Restricts Release Of Neutrophil Extracellular Traps

Background Immunothrombosis describes a process of hemostasis activation facilitates function of innate immune system,while immune system as participants of thrombosis.Increasing evidence suggest immunothrombosis as the key component of innate immune system in blood vessels during infection.Platelets and neutrophils plays as sentinels and first-line defendense during immunothrombosis,respectively.Moreover,serotonin is one of the most abundant substance derives from platelets.Neutrophils undergo NETosis leading to neutrophil extracellular traps(NETs)formation,eventually.This study focuses on influence of platelet-derived serotonin on NETs formation.Methods To explore the mechanisms and common factors associated with different type of NETs formation,we conducted global proteomics and phosphoproteomics analyses in neutrophils treated with phorbol 12-myristate 13-acetate(PMA),ionomycin,and monosodium urate(MSU).Furthermore,we conduncted experiments,both ex vivo and in vivo,on influence of platelet-derived serotonin on NETs formation.Results Global proteomic analyses identified 64,97,and 141 proteins differentially regulated in the PMA,ionomycin,and MSU groups compared with the control group,respectively.Phosphoproteomic analysis identified 931,565,and 201 phosphorylation sites differentially regulated in the PMA,ionomycin,and MSU groups,compared with the control,respectively.Overlap analysis of the three comparisons identified 9 proteins and 49 phosphorylation sites derived from 41 phosphoproteins.Among the 41 differentially regulated phosphoproteins,23 were associated with nuclear function,5 with chromatin binding,and 13 with poly(A)RNA binding activities based on GO annotation.Among these,DEK,methyl-CpG-binding protein 2(MECP2),and structure-specific recognition protein 1(SSRP1)are involved in both chromatin and poly(A)RNA binding.Ex vivo experiments demonstrated that serotonin inhibited PMA-induced NOX-dependent NETs via 5-HT2b activation.Similar results were obtained in ionomycin-induced NOX-independent NETs.In-depth study on mechanisms of NETs revealed that serotonin significantly reduced reactive oxygen species(ROS)production in early-phase PMA-induced NETs.In later-phase,serotonin alleviated degradation of histone and nuclear membrane induced by PMA,along with citrullination of histone.As stimulated time increased,PMA decreased phagocytic acivity of neutrophils,but serotonin managed to maintain some,meanwhile,switched neutrophil death from NETosis to apoptosis.Besides,serotonin also reduced ROS production induced by ionomycin.In vivo experiments of S.aureus-infected mouse model showed increased bacterial burden of liver,kidney,spleen and lung in mice treated with exogenous serotonin,along with absence of massive neutrophils aggregation in the liver.In experiments of cecal ligation and puncture mouse model,higher early phase mortality was found in serotonin transporter and tryptophan hydroxylase 1 knock-out mice.Conclusions Our study provides insight into molecular mechanisms of NETosis and a useful dataset for the guidance of future studies.Moreover,study of regulation of platelet on NETs provides insight of mechanisms underlying infection/sepsis.