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
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
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.
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.
