The Role Of Platelet-derived Exosomes In Neutrophil Extracellular Trap Formation During Sepsis-induced Acute Lung Injury

Acute lung injury(ALI)is a common manifestation of sepsis,characterized by high morbidity and mortality;however,the molecular mechanisms remain to be fully elucidated.Polymorphonuclear neutrophils(PMNs)are the primary effector cells of innate immunity during sepsis.Neutrophils release neutrophil extracellular traps(NETs)to enhance their antimicrobial properties.NETs are extracellular strands of decondensed DNA complexed with histones and neutrophil granule proteins.Although NETs exhibit protective roles during initial sepsis stages,excessive NET formation has been found to induce thrombosis and multiple organ failure in murine sepsis models.Platelets are not only elements of primary importance in hemostasis and thrombosis,but also essential elements of an integrated inflammatory response.Increasing evidence in vitro and in vivo indicates that platelets may contribute to the NET formation process by direct cell-to-cell contact and secreted substances.However,the mechanism underlying platelet-mediated NET formation remains unclear.Previous study has shown that platelet-derived exosomes sustain autophagy-associated NET formation in systemic sclerosis.However,whether platelet-derived exosomes play a role in NET formation in sepsis has yet to be addressed.Thus,this study aimed to detect whether platelet-derived exosomes could promote NET formation during sepsis and determine the potential mechanisms involved.Besides,we decided to find a possible target to improve ALI during sepsis through modulating platelet-derived exosome secretion.In order to address the aboved issues,this study was divided into four parts:The aim of the first part is to evaluate the value of NET as an indicator of sepsis.The results showed that the amounts of plasma ds DNA and MPO-DNA complexes were significantly higher in septic shock patients than those in healthy controls.Greater NET formation was associated with poor prognosis and positively correlated with SOFA scores in septic shock patients(r = 0.678,P = 0.0007).In addition,to investigate the role of platelets in sepsis-induced NET formation and ALI,platelets were depleted prior to conduct cecal ligation and puncture(CLP)model.Results showed that platelet depletion prior to CLP markedly decreased the NET formation,exosome concentration and lung injury.However,the bacterial load in the lungs was increased after platelet depletion.The second part is designed to detect whether platelet-derived exosomes could promote NET formation during sepsis and determine the potential mechanisms involved.The results showed that exosomes from LPS-stimulated platelets(LPS-Exo)induced NET formation in vitro,and LPS-Exo increased both ROS and autophagic activity in PMNs.However,NET formation was inhibited by autophagy inhibitors,but not by the ROS inhibitor after coculturing with LPS-Exo.Mammalian target of rapamycin(m TOR)is a dominant regulator of autophagy induction that inhibits autophagosome formation,and m TOR regulation by serine/threonine kinase Akt is well established.MHY1485-enhanced m TOR phosphorylation suppressed LPS-Exo induced NET formation.Moreover,phosphorylated m TOR(p-m TOR)and p-Akt levels were reduced after coculturing with LPS-Exo.Collectively,the above results indicated that platelet-derived exosomes increase autophagic activity in PMNs through modulating the Akt/m TOR pathway and eventually lead to greater NET formation during sepsis.The purpose of the third part is to address whether platelet-derived exosomes induce NET formation through HMGB1 and test the mi RNAs expression pattern in platelet-derived exosomes and find the key mi RNAs responsible for NET formation.First,we examined the presence of HMGB1 in exosomes by western blot and flow cytometry,and found a significantly higher HMGB1 expression level in LPS-Exo than in PBS-Exo.Autophagic activity and NET formation were inhibited in the presence of Box A,a HMGB1 antagonist.Next,we detected 84 differentially expressed mi RNAs.Enrichment pathway analysis showed that platelet activation and negative regulation of the PI3K/Akt network were within the 20 most enriched pathways.11 mi RNAs identified by mi RNA-seq could target the Akt/m TOR pathway and follow-up q RT-PCR verification showed that mi R-24-3p,mi R-15b-5p,mi R-25-3p,mi R-126-3p,mi R-378a-3p,and mi R-155-5p were significantly higher in sep-Exo than in con-Exo.Transfection of mi RNA mimics or inhibitors proved that platelet-derived exosomal mi R-15b-5p and mi R-378a-3p inhibited Akt/m TOR pathway activity by targeting PDK1 in PMNs,thus promoting NET formation.The aim of the fourth part is to determine the relationship between platelet-derived exosome secretion and IKK activity,and explore the possibility of using IKK inhibitors to improve ALI through decreasing platelet-derived exosome secretion.The results showed that IKK inhibitors effectively inhibited IκB phosphorylation and exosome secretion in LPS-stimulated platelets.The HMGB1 protein level in platelets after LPS stimulation was significantly decreased,and this effect was abrogated by IKK inhibitors.Besides,administration of IKK inhibitor(BMS-345541,10 mg/kg)to mice 2 h prior to CLP could decrease the plasma concentrations of exosomal protein,ds DNA and lung injury level.The above results indicated that platelet exosome secretion during sepsis was controlled by IKK.IKK may serve as a new target to alleviate ALI during sepsis.Conclusions: We demonstrated that platelet-derived exosomes containing HMGB1 and/or mi RNAs induce NET formation through modulating the Akt/m TORrelated autophagy pathway.The IKK inhibitor alleviates lung injury by inhibiting platelet-derived exosome secretion.These results indicate that signals associated with exosomes released from activated platelets serve as key features of sepsis.Targeting platelet-derived exosomes may present a novel therapeutic strategy for sepsis.