Macrophage Exosomes Promote Neutrophil Necroptosis Following Hemorrhagic Shock

Objective:Hemorrhagic shock（HS）,as a result of major trauma and severe surgery,causes an ischemia-reperfusion-type injury.Studies have suggested that HS can activate and prime the innate immune system to promote the development of multiple organ dysfunction syndrome（MOF）and systemic inflammatory response syndrome（SIRS）for an exaggerated inflammatory response and organ injury,while the mechanisms are still unclear.Macrophages and neutrophils are both important innate immune executive cells.Rather than acting as isolated effector cells,innate cells are in constant communication with other responding cells of the immune system.However,little is known about whether macrophages can regulate the PMN death response following HS.Besides,mounting evidence show that exosomes can mediate intercellular communications,stimulating target cells.In this study,we tested the hypothesis that HS acting through macrophage-derived exosomes alters PMN death response,thus induces PMN pro-inflammatory death,which enhances lung injury.Understanding these mechanisms may lead to improved interventions for surgical and trauma patients.Methods:1.Male C57BL/6 wild type（WT）mice and gp91 knockout(gp^91phox-/-)mice were randomly divided into sham operation group（Sham group）and hemorrhagic shock group（HS group）.Sham animals underwent the same surgical procedures without hemorrhagic and resuscitation.In some animals,at 24 h before performing hemorrhagic shock or sham operation,100 μl of clodrosome（Encapsula NanoSciences LLC）was injected intratracheally（i.t.）into the mice to deplete alveolar macrophages（AM?）.At 8 h after resuscitation,bronchoalveolar lavage（BAL）was performed and BAL fluid（BALF）was collected for experimental analysis.2.In order to recapitulate HS model in vitro,we used hypoxia/reoxygenation model to determine PMN death response to macrophages following hypoxia.PMN isolated from WT or gp^91phox-/-mice bone marrow were cocultured with BMDM,at 1:1 ratio in 1 ml of complete medium under different culture conditions.Hypoxia conditions were 1% O2,5% CO2,and 94% N2.After 15 h,hypoxic cultures were returned for reoxygenation in the normoxic incubator.Cells incubated under normoxic conditions（21% O2）served as controls.3.To determine PMN death response,the cell death rate was analyzed by flow cytometry double-positive staining with 7-AAD and Annexin V.To confirm the type of PMN death,cells were double stained with the RIPK1 and RIPK3.Colocalization of RIPK1 and RIPK3 was visualized by confocal microscopy and represents the RIPK1 and RIPK3 association to form the necrosome.Furthermore,immunoblotting examined the phosphorylation of RIPK1 and MLKL in PMN.4.Exosomes were isolated from culture supernatants using Total Exosome Isolation（from cell culture media）reagent.The average size of exosomes was determined by Nanoparticle Tracking Analysis.The amount of protein in exosomes was assessed by Lowry Protein Assay.The morphology of the exosomes was analyzed by transmission electron microscopy.The exosome marker protein CD63 was detected by flow cytometry.Results:1.Macrophages promote PMN necroptosis following hemorrhagic shock.Compared with sham group,the PMN 7-AAD and Annexin V double-positive rate of BALF was significant higher in HS group（P<0.01）.After AM? depletion by using clodrosome（i.t.）,the PMN 7-AAD and Annexin V double-positive rate was much lower than HS group（P<0.01）.Confocal microscopy shows the colocalization of RIPK1 and RIPK3 increased in HS group,after AM? depletion,the necrosome formation decreased than HS group（P<0.01）.2.Macrophages promote PMN necroptosis following hypoxia.After cocultured with BMDM under hypoxia condition,PMN cell death rate increased compared to other groups（P<0.01）,RIPK1 inhibitor necrostatin-1（Nec-1）could inhibit the increasement of cell death rate（P<0.01）.Western Blot shows the phosphorylation of RIPK1 and confocal microscopy shows the necrosome formation in PMN increased after cocultured with BMDM under hypoxia condition.Collectively,these results suggest that BMDM promote PMN death under hypoxia condition,and the portion of PMN death induced by BMDM under hypoxia condition is mainly necroptosis.3.Macrophages secret more exosomes following hemorrhagic shock.After isolation of exosomes from BALF,the expression of CD63 was detected using flow cytometry.The result shows that the expression of CD63 increased following HS（P<0.01）,while depletion of AM? could prevent the increase of CD63 expression following HS（P<0.01）.Transmission electron microscopy shows the cup-shaped morphology of exosomes isolated from BALF.4.Macrophages secret more exosomes following hemorrhagic shock.After isolation of exosomes from cell culture medium,the size was analyzed by using NTA and the average size of exosomes was 39.86 nm.The protein level of exosomes isolated from BMDM medium under hypoxia condition was about 4 folds of that under normoxia condition（P<0.01）,and the expression of exosomes marker protein CD63 was also significantly higher in hypoxia-conditoned BMDM medium as compared to normoxia-conditioned BMDM medium（P=0.009）.Besides,after cocultured with dil-labeled BMDM exosomes,confocal microscopy shows the internalization of exosomes in PMN.5.Exosome-releasing inhibitor inhibits PMN necroptosis promtoted by macrophages following hypoxia.After pretreated with exosome-releasing inhibitor DMA,PMN necroptosis and the phosphorylation of RIPK1 and MLKL in PMN were prevented.Confocal microscopy also shows that DMA attenueted the necrosome formation in PMN.（P<0.01）6.Macrophages promote PMN necroptosis through exosomes secretion following hypoxia.Flow cytometry shows that,after cocultured with BMDM-derived exosomes from hypoxia condition,the necroptosis of PMN from WT mice increased and could be prevented by Nec-1（P<0.01）.7.Macrophage-derived exosomes upregulated PMN ROS level following hypoxia.In hypoxia and coculture group,the ROS level in PMN was significantly higher as compared to other groups（P<0.01）,and could be prevented by DMA（P<0.01）.Besides,BMDM-derived exosomes from hypoxia condition upregulated the ROS level in PMN from WT mice（P<0.01）.8.Macrophage-derived exosomes promote PMN necroptosis through upregulating PMN ROS level following hypoxia.After pretreated with ROS NAC,BMDM couldn’t promote PMN necroptosis following hypoxia.BMDM from WT mice didn’t induce necroptosis and necrosome formation in PMN from gp^91phox-/-mice,while BMDM from gp^91phox-/-mice still could induce necroptosis and necrosome formation in PMN from WT mice.Besides,BMDM-derived exosomes from hypoxia condition couldn’t promote the necroptosis of PMN from gp^91phox-/-mice and upregulate the ROS level in gp^91phox-/-PMN（P<0.01）.9.NADPH oxidase plays an important role in promoting PMN necroptosis following hemorrhagic shock.PMN necroptosis and necrosome formation in PMN from gp^91phox-/-mice decreased following HS as compared to that from WT mice（P<0.01）.Conclusion:Hemorrhagic shock induces AMφ-derived exosomes secretion.Therefore,the macrophage-derived exosomes induce ROS,which source from NADPH oxidase,increase in PMN.Thus,the exosomes from AM? promote neutrophil necroptosis,which further amplified the lung injury during hemorrhagic shock.