The Effect And Mechanism Of Microglia-derived Small Extracellular Vesicles On Macrophages In Traumatic Brain Injury

Background:Traumatic brain injury(TBI)is a major global health concern,leading to numerous deaths and disabilities annually.Following TBI,the neuroinflammatory response plays a crucial role in secondary brain injury.As the brain’s innate immune cells,microglia sense injury-associated molecular patterns(DAMPs)after brain injury and recruit peripheral immune cells through a series of signaling pathways,contributing collectively to brain repair.Monocytes/macrophages are among the primary cells recruited after the blood-brain barrier becomes permeable,playing key roles in phagocytosis of cellular debris,pathogen clearance,and tissue repair,significantly influencing the prognosis of TBI.Studies show that the neuroinflammatory response is closely involved and decisive in the prognosis of TBI.After TBI,microglia are activated and release chemokines to recruit a large number of inflammatory monocytes into the damaged brain tissue,which is a key factor in inducing neuroinflammatory responses.Recent research has begun to unveil the complex roles of microglia and macrophages in the inflammatory response following TBI.While microglia are primarily considered to recruit myeloid monocytes/macrophages through the secretion of chemokine CCL2,other communication methods such as exosomes also play roles in this process,though there is no consensus yet.Therefore,studying the communication parttern between microglia and macrophages after TBI,and the role and mechanism of their secreted small extracellular vesicles(sEVs)in recruiting and influencing macrophages,is crucial for understanding the post-TBI neuroinflammatory response and for developing new therapies targeting neuroinflammation.Purpose:(1)Explore the modes of communication between microglia and macrophages during the acute phase of TBI in mice and their impact on the progression of neuroinflammation.(2)Explore the release mechanisms of sEVs derived from inflammatory microglia,the patterns of internalization by macrophages,and their effects on macrophages.(3)Explore the mechanisms by which sEVs from inflammatory microglia promote macrophage activation.(4)Explore the roles of sEVs from inflammatory microglia in neuroinflammation,neurological function recovery,and histological repair in TBI mice.Methods:Part One: Firstly,a TBI neuroinflammation mouse model is established through cortical puncture surgery.Secondly,at different time points during the acute phase of TBI,methods such as blood cell analysis,frozen section staining,and flow cytometry are used to detect and analyze the number and activation states of peripheral blood mononuclear cells,microglia in the injured area,and mononuclear macrophages.Then,the local and systemic expression of inflammatory cytokines at the injury site is analyzed using flow cytometric bead array assays.Finally,single-cell sequencing technology is utilized to analyze the communication and gene expression relationships between microglia and macrophages during the acute phase of TBI.Part Two: Firstly,a Transwell model is established between microglial BV2 cells and macrophage RAW264.7 cells to observe whether GW4869 inhibition of sEVs secretion affects macrophage migration.Secondly,the optimal lipopolysaccharide(LPS)concentration for inflammatory activation of microglia is determined through CCK-8 assay,morphological staining,and q PCR experiments.Thirdly,sEVs derived from microglia are isolated using ultracentrifugation,and characterized by Western blotting,transmission electron microscopy,nano-flow cytometry,Nanosight,BCA protein quantification,and total protein staining.Then,macrophage phagocytosis is assessed after labeling sEVs with PKH67,using flow cytometry and confocal imaging.Finally,changes in activation status and phagocytic migration ability of macrophages after internalizing sEVs are analyzed through light microscopy,CD86 staining flow cytometry,fluorescent latex bead uptake,and Transwell assays.Part Three: Firstly,the proteomic composition of sEVs derived from microglia is analyzed using mass spectrometry.Secondly,transcriptional changes in macrophages that have phagocytosed sEVs are analyzed through RNA-seq.Then,the presence of LPS in sEVs is tested using Limulus amoebocyte lysate assay.Finally,the expression of certain pathway and surface marker proteins is verified using Western blot and immunofluorescence staining.Part Four: Firstly,two hours post-TBI,sEVs derived from in vitro cultured microglia are injected into mice via the tail vein,and twelve hours post-injury,the number and activation of mononuclear macrophages in peripheral blood and injured brain tissue,as well as the expression levels of inflammatory cytokines,are detected using flow cytometry.Secondly,a moderate to severe TBI mouse model is established using fluid percussion injury.Then,similarly,two hours post-TBI,sEVs are injected into mice via the tail vein,and motor and learning memory abilities are assessed using the rotarod test,open field test,novel object recognition test,and water maze test.Finally,sixteen days post-TBI,mouse brain tissue is assessed histologically using frozen section immunofluorescence staining.Results:Part One: Blood cell analysis showed that,8-16 hours post-TBI,the number of peripheral blood mononuclear cells peaked.Nissl staining indicated that secondary neuronal damage began to intensify after 16 hours.Immunostaining revealed that macrophage-lineage cells were widely activated at 48 and 72 hours post-injury.Flow cytometry demonstrated that the number of microglia at the injury site peaked at 6 hours post-injury,while the number of macrophages continued to increase during the acute phase;microglial activation peaked at 24 hours,whereas macrophage activation continued to rise.Inflammation marker analysis showed that systemic inflammatory response was mild,but the expression of inflammatory markers at the injury site peaked 12 hours post-injury.Single-cell sequencing revealed multiple communication pathways between microglia and macrophages,including Ptprc-Mrc1,App-Cd74,and Mif-Cd74+Cd44.Twelve hours post-injury,their functions were enriched in cellular components related to exosome production,and by 48 hours post-injury,the degree of KEGG pathway enrichment tended to converge.Additionally,macrophages recruited during the acute phase highly expressed anti-inflammatory reparative macrophage genes such as Arg-1.Part Two: Cell migration experiments found that LPS-activated microglia could regulate macrophage migration ability,and this regulation depended on sEVs secretion to a certain extent.sEVs were successfully isolated and identified,with inflammatory microglial sEVs showing an increased average diameter and a new population peak at189 nm,lower yields,and differential protein expression.PKH67 labeling experiments revealed that inflammatory microglial sEVs were more readily internalized by macrophages.Macrophages that phagocytosed inflammatory microglial sEVs differentiated into an amoeboid morphology,showed increased cell body size,higher CD86 expression,and significantly enhanced phagocytosis and migration abilities.Also,macrophages that internalized inflammatory microglial sEVs showed significantly enhanced expression of TNF-α,i NOS genes,and significant increases in inflammatory protein secretion of IL-1β,IL-6,IL-10,CCL2,and TNF.Part Three: Proteomic sequencing revealed that differential proteins in inflammatory microglial sEVs were primarily increased,with the highest fold change in the autophagy-related protein Esyt1.IPA analysis indicated that differential proteins were mainly enriched in immune inflammation-related collections.Macrophage sequencing revealed significant differences in gene expression in the inflammatory sEVs group,with the most significant differences in genes such as Cxcl2,Acod1,Csf3,Ccl3,and Ccl2.Bioinformatics analysis showed that the inflammatory sEVs group was enriched in KEGG pathways such as the TNF pathway,cytokine-cytokine receptor interaction,IL-17 signaling pathway,NF-κB signaling pathway,and GO processes like ERK1 and ERK2 cascade,LPS-mediated cell response,immune response,inflammatory response,and bacterial response.Endotoxin testing with Limulus amebocyte lysate assay ruled out inflammatory signal transmission due to LPS contamination in sEVs.Western blot techniques confirmed increased IRAK4 in the NOD-like pathway,increased polarization proteins IRF5,CD16,Arg-1,increased nuclear p65,p-p65 in the NF-κB pathway,decreased cytoplasmic p65,p-iκBα,increased cytoplasmic p-p65,and increased LC3 BII,p62,decreased FAM134 B in the autophagy pathway for macrophages that phagocytosed inflammatory sEVs.Part Four: Flow cytometry revealed that inflammatory microglial sEVs significantly increased the number of mononuclear macrophages in peripheral blood 12 hours post-TBI without affecting polarization and significantly increased the number and elevated the CD86 polarization level of mononuclear macrophages at the brain injury site.Inflammatory factor analysis found that inflammatory microglial sEVs caused increases in IL-10,TNF,CCL2 in peripheral blood and IL-1β,IL-6,TNF,CCL2 at the injury site 12 hours post-TBI.Behavioral tests showed that inflammatory microglial sEVs significantly improved mouse performance in the rotarod test for motor balance,new object recognition ability,and learning and memory ability in the water maze test.Brain tissue section immunostaining found that inflammatory microglial sEVs reduced the volume of brain injury lesions,reduced the density of microglia and astrocytes at the injury site,and increased capillary formation and neuronal regeneration.Conclusion:(1)During the early acute phase of TBI,microglia primarily propagate inflammatory signals,while in the later stages,they mainly undertake phagocytosis and repair functions;monocyte-derived macrophages mainly exhibit anti-inflammatory and reparative functions during the acute phase;there exists cellular communication between microglia and macrophages,and this communication may be partly mediated by extracellular vesicles.(2)When stimulated by inflammation,microglia can transmit inflammatory signals through sEVs,specifically activating macrophage phagocytosis,migration,and paracrine secretion abilities.(3)The possible mechanism through which microglia activate macrophages via sEVs involves the transfer of inflammatory and autophagy-related proteins by sEVs,reducing macrophage autophagy levels,and activating inflammation-related pathways.(4)Inflammatory microglial sEVs can enhance the function of mononuclear macrophages during the acute phase of mouse TBI and improve the local inflammatory environment,while also promoting the recovery of motor and learning memory abilities during the subacute phase,reducing glial cell activation in the chronic phase,and promoting vascular and neuronal regeneration.This study will provide a better understanding of the pathological process and mechanism of TBI,and provide a new target for the treatment of secondary neuroinflammatory response after TBI.