Background:Inflammation is an important factor in pathological scarring.The role of neutrophils,one of the most important inflammatory cells,in scar hyperplasia remains unclear.The purpose of this article is to study ...Background:Inflammation is an important factor in pathological scarring.The role of neutrophils,one of the most important inflammatory cells,in scar hyperplasia remains unclear.The purpose of this article is to study the correlation between neutrophil extracellular traps(NETs)and scar hyperplasia and identify a new target for inhibiting scar hyperplasia.Methods:Neutrophils were isolated from human peripheral blood by magnetic-bead sorting.NETs in plasma and scars were detected by enzyme-linked immunosorbent assays(ELISAs),immunofluorescence and flow cytometry.Immunohistochemistry was used to assess neutrophil(CD66B)infiltration in hypertrophic scars.To observe the entry of NETs into fibroblasts we used immunofluorescence and flow cytometry.Results:We found that peripheral blood neutrophils in patients with hypertrophic scars were more likely to form NETs(p<0.05).Hypertrophic scars showed greater infiltration with neutrophils and NETs(p<0.05).NETs activate fibroblasts in vitro to promote their differentiation and migration.Inhibition of NETs with cytochalasin in wounds reduced the hyperplasia of scars in mice.We induced neutrophils to generate NETs with different stimuli in vitro and detected the proteins carried by NETs.We did not find an increase in the expression of common scarring factors[interleukin(IL)-17 and transforming growth factor-β(TGF-β),p>0.05].However,inhibiting the production of NETs or degrading DNA reduced the differentiation of fibroblasts intomyofibroblasts.In vitro,NETs were found to be mediated by Toll-like receptor 9(TLR-9)in fibroblasts and further phosphorylated nuclear factor Kappa-B(NF-κB).We found that IL-6,which is downstream of NF-κB,was increased in fibroblasts.Additionally,IL-6 uses autocrine and paracrine signaling to promote differentiation and secretion.Conclusions:Our experiments found that NETs activate fibroblasts through the TLR-9/NF-κB/IL-6 pathway,thereby providing a new target for regulating hypertrophic scars.展开更多
Background:Burn shock caused by vascular leakage is one of the main causes of high mortality in severe burn injury.However,the pathophysiological mechanism of vascular leakage is still unclear.The purpose of this stud...Background:Burn shock caused by vascular leakage is one of the main causes of high mortality in severe burn injury.However,the pathophysiological mechanism of vascular leakage is still unclear.The purpose of this study was to explore the molecular mechanism of vascular leakage in the early stage of severe burn and provide a new target for the treatment of severe burns.Methods:Neutrophils were isolated from human peripheral blood by magnetic beads sorting.ELISA was used to detect neutrophil-derived granule proteins and glycocalyx injury products in plasma.The vascular leakage and neutrophil movement were assessed by in vivo laser confocal imaging in mice,and high-quality video were provided.Adhesion-related molecules were investigated by qRT-PCR.The damage to glycocalyx of mice vascular endothelial cellswas observed by transmission electron microscope and scanning electron microscope.Proteomic analysis,flow cytometry and immunofluorescence were used to further study the relationship between human peripheral blood neutrophil-derived hypochlorite(HOCl)and CD44 of human vascular endothelial cells.Results:In this study,we found that rapidly increasing activated neutrophils secrete heparin binding protein(HBP)andmyeloperoxidase(MPO)after severe burn injury.Increased HBP triggers vascular leakage with synergy of MPO,results in systemic edema and burn shock.Furthermore,we found that the MPO catalytic product HOCl but not MPO triggers CD44 extracellular domain shedding from vascular endothelial cells to damage the glycocalyx.Damage to the glycocalyx results in firm adhesion of neutrophils and increases vascular leakage.However,MPO inhibitors partially protect the glycocalyx of vascular endothelial cells.The combination of HBP and MPO inhibitors markedly reduces vascular leakage and systemic edema in the early stage of severe burns.Conclusions:Taken together,these data reveal that neutrophil-derived HBP and MPO play an important synergies role in triggering vascular leakage at the early stage of severe burns.Targeted intervention in these two biomolecules may introduce new strategies for helping to reduce large amount of fluid loss and subsequent burn shock.展开更多
Autophagy,an adaptive catabolic process,plays a cytoprotective role in enabling cellular homeostasis in the innate and adaptive immune systems.Neutrophils,the most abundant immune cells in circulation,are professional...Autophagy,an adaptive catabolic process,plays a cytoprotective role in enabling cellular homeostasis in the innate and adaptive immune systems.Neutrophils,the most abundant immune cells in circulation,are professional killers that orchestrate a series of events during acute inflammation.The recent literature indicates that autophagy has important roles in regulating neutrophil functions,including differentiation,degranulation,metabolism and neutrophil extracellular trap formation,that dictate neutrophil fate.It is also becoming increasingly clear that autophagy regulation is critical for neutrophils to exert their immunological activity.However,evidence regarding the systematic communication between neutrophils and autophagy is insufficient.Here,we provide an updated overview of the function of autophagy as a regulator of neutrophils and discuss its clinical relevance to provide novel insight into potentially relevant treatment strategies.展开更多
Background:Early detection,timely diagnosis and rapid response are essential for case management and precautions of burn-associated sepsis.However,studies on indicators for early warning and intervention have rarely b...Background:Early detection,timely diagnosis and rapid response are essential for case management and precautions of burn-associated sepsis.However,studies on indicators for early warning and intervention have rarely been conducted.This study was performed to better understand the pathophysiological changes and targets for prevention of severe burn injuries.Methods:We conducted a multi-center,prospective multi-omics study,including genomics,microRNAomics,proteomics and single-cell transcriptomics,in 60 patients with severe burn injuries.A mouse model of severe burn injuries was also constructed to verify the early warning ability and therapeutic effects of potential markers.Results:Through genomic analysis,we identified seven important susceptibility genes(DNAH11,LAMA2,ABCA2,ZFAND4,CEP290,MUC20 and ENTPD1)in patients with severe burn injuries complicated with sepsis.Through plasma miRNAomics studies,we identified four miRNAs(hsamiR-16-5p,hsa-miR-185-5p,hsa-miR-451a and hsa-miR-423-5p)that may serve as early warning markers of burn-associated sepsis.A proteomic study indicated the changes in abundance of major proteins at different time points after severe burn injury and revealed the candidate early warning markers S100A8 and SERPINA10.In addition,the proteomic analysis indicated that neutrophils play an important role in the pathogenesis of severe burn injuries,as also supported by findings from single-cell transcriptome sequencing of neutrophils.Through further studies on severely burned mice,we determined that S100A8 is also a potential early therapeutic target for severe burn injuries,beyond being an early warning indicator.Conclusions:Our multi-omics study identified seven susceptibility genes,four miRNAs and two proteins as early warning markers for severe burn-associated sepsis.In severe burn-associated sepsis,the protein S100A8 has both warning and therapeutic effects.展开更多
Neutrophil and platelet are essential arms of the innate immune response. In sepsis, platelet abnormal activation as well as neu-trophil paralysis are well recognized. For platelet, it is characterized by the contribu...Neutrophil and platelet are essential arms of the innate immune response. In sepsis, platelet abnormal activation as well as neu-trophil paralysis are well recognized. For platelet, it is characterized by the contribution to disseminated intravascular coagulation (DIC) and the enhanced inflammation response. In terms of neutrophil, its dysfunction is manifested by the impaired recruitment and migration to the infectious foci, abnormal sequestration in the remote organs, and the delayed clearance. More recently, it has been apparent that together platelet-neutrophil interaction can induce a faster and harder response during sepsis. This article focuses on the activation of platelet, dysfunction of neutrophil, and the interaction between them during sepsis and profiles some of the molecular mechanisms and outcomes in these cellular dialogues, providing a novel strategy for treatment of sepsis.展开更多
Background:Bacterial infections pose a considerable threat to skin wounds,particularly in the case of challenging-to-treat diabetic wounds.Systemic antibiotics often struggle to penetrate deep wound tissues and topica...Background:Bacterial infections pose a considerable threat to skin wounds,particularly in the case of challenging-to-treat diabetic wounds.Systemic antibiotics often struggle to penetrate deep wound tissues and topically applied antibiotics may lead to sensitization,necessitating the development of novel approaches for effectively treating germs in deep wound tissues.Neutrophils,the predominant immune cells in the bloodstream,rapidly release an abundance of molecules via degranulation upon activation,which possess the ability to directly eliminate pathogens.This study was designed to develop novel neutrophil cell engineered nanovesicles(NVs)with high production and explore their bactericidal properties and application in promoting infectious wound healing.Methods:Neutrophils were isolated from peripheral blood and activated in vitro via phorbol myristate acetate(PMA)stimulation.Engineered NVs were prepared by sequentially extruding activated neutrophils followed by ultracentrifugation and were compared with neutrophil-derived exosomes in terms of morphology,size distribution and protein contents.The bactericidal effect of NVs in vitro was evaluated using the spread plate technique,LIVE/DEAD backlight bacteria assay and observation of bacterial morphology.The therapeutic effects of NVs in vivo were evaluated using wound contraction area measurements,histopathological examinations,assessments of inflammatory factors and immunochemical staining.Results:Activated neutrophils stimulated with PMA in vitro promptly release a substantial amount of bactericidal proteins.NVs are similar to exosomes in terms of morphology and particle size,but they exhibit a significantly higher enrichment of bactericidal proteins.In vitro,NVs demonstrated a significant bactericidal effect,presumably mediated by the enrichment of bactericidal proteins such as lysozyme.These NVs significantly accelerated wound healing,leading to a marked reduction in bacterial load,downregulation of inflammatory factors and enhanced collagen deposition in a fullthickness infectious skin defect model.Conclusions:We developed engineered NVs derived from activated neutrophils to serve as a novel debridement method targeting bacteria in deep tissues,ultimately promoting infectious wound healing.展开更多
基金supported by the National Natural Science Foundation of China,(No.82072217,81772135 and U21A20370)by the Jiangsu Natural Science Foundation(No.BK20201178).
文摘Background:Inflammation is an important factor in pathological scarring.The role of neutrophils,one of the most important inflammatory cells,in scar hyperplasia remains unclear.The purpose of this article is to study the correlation between neutrophil extracellular traps(NETs)and scar hyperplasia and identify a new target for inhibiting scar hyperplasia.Methods:Neutrophils were isolated from human peripheral blood by magnetic-bead sorting.NETs in plasma and scars were detected by enzyme-linked immunosorbent assays(ELISAs),immunofluorescence and flow cytometry.Immunohistochemistry was used to assess neutrophil(CD66B)infiltration in hypertrophic scars.To observe the entry of NETs into fibroblasts we used immunofluorescence and flow cytometry.Results:We found that peripheral blood neutrophils in patients with hypertrophic scars were more likely to form NETs(p<0.05).Hypertrophic scars showed greater infiltration with neutrophils and NETs(p<0.05).NETs activate fibroblasts in vitro to promote their differentiation and migration.Inhibition of NETs with cytochalasin in wounds reduced the hyperplasia of scars in mice.We induced neutrophils to generate NETs with different stimuli in vitro and detected the proteins carried by NETs.We did not find an increase in the expression of common scarring factors[interleukin(IL)-17 and transforming growth factor-β(TGF-β),p>0.05].However,inhibiting the production of NETs or degrading DNA reduced the differentiation of fibroblasts intomyofibroblasts.In vitro,NETs were found to be mediated by Toll-like receptor 9(TLR-9)in fibroblasts and further phosphorylated nuclear factor Kappa-B(NF-κB).We found that IL-6,which is downstream of NF-κB,was increased in fibroblasts.Additionally,IL-6 uses autocrine and paracrine signaling to promote differentiation and secretion.Conclusions:Our experiments found that NETs activate fibroblasts through the TLR-9/NF-κB/IL-6 pathway,thereby providing a new target for regulating hypertrophic scars.
基金supported by the National Natural Science Founda-tion of China,No.82072217,81772135the Jiangsu Natural Science Foundation,No.BK20201178。
文摘Background:Burn shock caused by vascular leakage is one of the main causes of high mortality in severe burn injury.However,the pathophysiological mechanism of vascular leakage is still unclear.The purpose of this study was to explore the molecular mechanism of vascular leakage in the early stage of severe burn and provide a new target for the treatment of severe burns.Methods:Neutrophils were isolated from human peripheral blood by magnetic beads sorting.ELISA was used to detect neutrophil-derived granule proteins and glycocalyx injury products in plasma.The vascular leakage and neutrophil movement were assessed by in vivo laser confocal imaging in mice,and high-quality video were provided.Adhesion-related molecules were investigated by qRT-PCR.The damage to glycocalyx of mice vascular endothelial cellswas observed by transmission electron microscope and scanning electron microscope.Proteomic analysis,flow cytometry and immunofluorescence were used to further study the relationship between human peripheral blood neutrophil-derived hypochlorite(HOCl)and CD44 of human vascular endothelial cells.Results:In this study,we found that rapidly increasing activated neutrophils secrete heparin binding protein(HBP)andmyeloperoxidase(MPO)after severe burn injury.Increased HBP triggers vascular leakage with synergy of MPO,results in systemic edema and burn shock.Furthermore,we found that the MPO catalytic product HOCl but not MPO triggers CD44 extracellular domain shedding from vascular endothelial cells to damage the glycocalyx.Damage to the glycocalyx results in firm adhesion of neutrophils and increases vascular leakage.However,MPO inhibitors partially protect the glycocalyx of vascular endothelial cells.The combination of HBP and MPO inhibitors markedly reduces vascular leakage and systemic edema in the early stage of severe burns.Conclusions:Taken together,these data reveal that neutrophil-derived HBP and MPO play an important synergies role in triggering vascular leakage at the early stage of severe burns.Targeted intervention in these two biomolecules may introduce new strategies for helping to reduce large amount of fluid loss and subsequent burn shock.
基金supported by the National Natural Science Foundation of China(No.81772135,No.81471903)the Jiangsu Natural Science Foundation(No.BE2017695).
文摘Autophagy,an adaptive catabolic process,plays a cytoprotective role in enabling cellular homeostasis in the innate and adaptive immune systems.Neutrophils,the most abundant immune cells in circulation,are professional killers that orchestrate a series of events during acute inflammation.The recent literature indicates that autophagy has important roles in regulating neutrophil functions,including differentiation,degranulation,metabolism and neutrophil extracellular trap formation,that dictate neutrophil fate.It is also becoming increasingly clear that autophagy regulation is critical for neutrophils to exert their immunological activity.However,evidence regarding the systematic communication between neutrophils and autophagy is insufficient.Here,we provide an updated overview of the function of autophagy as a regulator of neutrophils and discuss its clinical relevance to provide novel insight into potentially relevant treatment strategies.
基金supported by the National Natural Science Foundation of China,No.82072217,81772135 and U21A20370the Jiangsu Natural Science Foundation,No.BE2017695 and BK20201178.
文摘Background:Early detection,timely diagnosis and rapid response are essential for case management and precautions of burn-associated sepsis.However,studies on indicators for early warning and intervention have rarely been conducted.This study was performed to better understand the pathophysiological changes and targets for prevention of severe burn injuries.Methods:We conducted a multi-center,prospective multi-omics study,including genomics,microRNAomics,proteomics and single-cell transcriptomics,in 60 patients with severe burn injuries.A mouse model of severe burn injuries was also constructed to verify the early warning ability and therapeutic effects of potential markers.Results:Through genomic analysis,we identified seven important susceptibility genes(DNAH11,LAMA2,ABCA2,ZFAND4,CEP290,MUC20 and ENTPD1)in patients with severe burn injuries complicated with sepsis.Through plasma miRNAomics studies,we identified four miRNAs(hsamiR-16-5p,hsa-miR-185-5p,hsa-miR-451a and hsa-miR-423-5p)that may serve as early warning markers of burn-associated sepsis.A proteomic study indicated the changes in abundance of major proteins at different time points after severe burn injury and revealed the candidate early warning markers S100A8 and SERPINA10.In addition,the proteomic analysis indicated that neutrophils play an important role in the pathogenesis of severe burn injuries,as also supported by findings from single-cell transcriptome sequencing of neutrophils.Through further studies on severely burned mice,we determined that S100A8 is also a potential early therapeutic target for severe burn injuries,beyond being an early warning indicator.Conclusions:Our multi-omics study identified seven susceptibility genes,four miRNAs and two proteins as early warning markers for severe burn-associated sepsis.In severe burn-associated sepsis,the protein S100A8 has both warning and therapeutic effects.
基金National Natural Science Foundation of China, No. 30772256, 81071546 and No. 81272148, and Jiangsu Provincial Natural Science Founda-tion, No. BK2012703
文摘Neutrophil and platelet are essential arms of the innate immune response. In sepsis, platelet abnormal activation as well as neu-trophil paralysis are well recognized. For platelet, it is characterized by the contribution to disseminated intravascular coagulation (DIC) and the enhanced inflammation response. In terms of neutrophil, its dysfunction is manifested by the impaired recruitment and migration to the infectious foci, abnormal sequestration in the remote organs, and the delayed clearance. More recently, it has been apparent that together platelet-neutrophil interaction can induce a faster and harder response during sepsis. This article focuses on the activation of platelet, dysfunction of neutrophil, and the interaction between them during sepsis and profiles some of the molecular mechanisms and outcomes in these cellular dialogues, providing a novel strategy for treatment of sepsis.
基金supported by National Natural Science Foundation of China(No.82072217,81772135 and U21A20370).
文摘Background:Bacterial infections pose a considerable threat to skin wounds,particularly in the case of challenging-to-treat diabetic wounds.Systemic antibiotics often struggle to penetrate deep wound tissues and topically applied antibiotics may lead to sensitization,necessitating the development of novel approaches for effectively treating germs in deep wound tissues.Neutrophils,the predominant immune cells in the bloodstream,rapidly release an abundance of molecules via degranulation upon activation,which possess the ability to directly eliminate pathogens.This study was designed to develop novel neutrophil cell engineered nanovesicles(NVs)with high production and explore their bactericidal properties and application in promoting infectious wound healing.Methods:Neutrophils were isolated from peripheral blood and activated in vitro via phorbol myristate acetate(PMA)stimulation.Engineered NVs were prepared by sequentially extruding activated neutrophils followed by ultracentrifugation and were compared with neutrophil-derived exosomes in terms of morphology,size distribution and protein contents.The bactericidal effect of NVs in vitro was evaluated using the spread plate technique,LIVE/DEAD backlight bacteria assay and observation of bacterial morphology.The therapeutic effects of NVs in vivo were evaluated using wound contraction area measurements,histopathological examinations,assessments of inflammatory factors and immunochemical staining.Results:Activated neutrophils stimulated with PMA in vitro promptly release a substantial amount of bactericidal proteins.NVs are similar to exosomes in terms of morphology and particle size,but they exhibit a significantly higher enrichment of bactericidal proteins.In vitro,NVs demonstrated a significant bactericidal effect,presumably mediated by the enrichment of bactericidal proteins such as lysozyme.These NVs significantly accelerated wound healing,leading to a marked reduction in bacterial load,downregulation of inflammatory factors and enhanced collagen deposition in a fullthickness infectious skin defect model.Conclusions:We developed engineered NVs derived from activated neutrophils to serve as a novel debridement method targeting bacteria in deep tissues,ultimately promoting infectious wound healing.
