Regulatory Effects And Mechanisms Of Neutrophil Extracellular Traps On Brain Pericytes In Traumatic Brain Injury

Traumatic brain injury(TBI)is a major public health issue worldwide and one of the leading causes of physical disability and death for young people.The injury that occurs in TBI can be separated into a primary injury and a delayed secondary injury involving a variety of changes at the cellular and molecular levels.The initial injury emerges immediately at the moment of impact and is a mechanical damage of neurons,axons and blood vessels resulting from an acceleration-deceleration mechanism.In the following hours to days,the secondary injury evolves as a result of neuronal and glial dysfunction,metabolic changes,cerebral edema and neuroinflammation.The secondary injury is the main pathological factors to trigger neurological deficits and dysfunctions,providing potential targets for therapeutic interventions.Neuroinflammation plays an important role in the pathogenesis of the secondary injury,which involves a complex array of molecular biological mechanisms.The inflammation after TBI is participated by both central immune cells(microglias,astrocytes and immune cells in cerebral lymphatic vessels)and peripheral immune cells.Neutrophils are the most abundant innate immune cells in the peripheral circulation.They are typically the first leukocytes infiltrating to the cerebral inflammatory sites after TBI and perform their function by phagocytosis and degranulation to clear tissue debris and pathogens.In addition,there is a novel and special antimicrobial mechanism of neutrophil,which is referred to as neutrophil extracellular traps(NETs).The NETs formation may contribute to the disruption of BBB integrity and lead to neurological damage after ischemic stroke.While,the roles and possible mechanisms of NETs post TBI are largely unknown.The blood brain barrier(BBB)is comprised of vascular basement membranes,endothelial cells,astrocytes and pericytes.The tight junctions between adjacent endothelial cells are the primary morphological basis for the BBB.Pericytes are centrally positioned between endothelial cells,astrocytes,and neurons.They receive and integrate signals from their neighboring cells and generate functional responses that are essential for proper regulation of the central nervous system.Pericytes in BBB have diverse functions,including cerebral angiogenesis,BBB permeability regulation,removal of toxic metabolites and neuroinflammation regulation.Pericytes are able to chemo-attract neutrophils via secreting chemokines under neurological or inflammatory conditions,while NETs formation can disrupt the BBB and increase BBB permeability,thus aggravating neuroinflammation.However,the role of NETs and its interaction with pericytes after TBI are still poorly understood.Therefore,this thesis mainly focused on the interaction between NETs and pericytes in the BBB disruption after TBI and the underlying mechanisms,expecting to provide potential therapeutic target for the recovery of neurological function.Section 1:Phenotypic changes and functional characteristic of brain pericytes after TBIBrain pericytes exhibit great plasticity in certain diseases state and.the disruption of BBB after TBI is accompanied by the loss of brain pericytes.At present,the phenotypic changes and functional characteristic of brain pericytes after TBI have not been reported.To observe the phenotypic change of brain pericytes after TBI,pericytes were firstly sorted respectively from TBI mice and sham-operated mice for RNA-Sequencing.The RNA-Seq indicated that the expression of several genes was changed after TBI.Among these genes,we focused on ITGAM(CD11b-encoding gene),which was predominantly detected in neutrophils,monocytes,macrophages and microglia.It was previously reported that brain pericytes could expressed CD11b under hypoxia or inflammatory conditions.However,whether CD11b expression could be observed in pericytes after TBI has not yet be reported.Using immunofluorescence,we confirmed the existence of CD11b~+pericytes in brain tissues from TBI patients and TBI mice.The FC results showed that CD11b~+pericytes were significantly increased in damaged brain after TBI compared to Sham.In order to explore the differences between CD11b~+and CD11b~-pericytes,we sorted these subpopulations from the damaged brains of TBI mice and prepared them for RNA-sequencing,and the RNA-seq showed that the m RNA levels of several pro-inflammatory genes(TNF-α,IL-1β,CCL5,et al.)and BBB disruption-related genes(MMP2 and MMP9)in CD11b~+pericytes were significantly higher than in CD11b~-pericytes,which was validated by q RT-PCR.These data provide evidence that the upregulation of CD11b was one of the phenotypic changes in brain pericytes after TBI,and these CD11b~+pericytes were possibly important contributors to neuroinflammation and BBB disruption in the pathogenesis of TBI.Section 2:The NETs formation after TBI and its effects on brain pericytesNeutrophils were isolated from peripheral blood of TBI mice and sham-operated mice,and these neutrophils were cultured with PMA stimulation.The results showed that neutrophils from TBI mice formed NETs more easily post stimulation.FC experiments further showed that the percentage of NETs formation in the core lesion area was relatively higher than the para-injury area,the contralateral side and the sham mice.Meanwhile,the neutrophil infiltration rate was positively associated with the proportion of CD11b~+pericytes in damaged brain.Thus,we speculated that NETs formation was possibly related to CD11b induction on pericytes.To test this hypothesis,we treated Mouse Brain Vascular Pericytes(MBVP)with NETs-induced medium(under stimulation with PMA).After culturing with NETs-induced medium for 48h,FC analysis showed that CD11b expression was strongly induced in MBVP.Immunofluorescence,western blotting,and q RT-PCR experiments also supported the conclusion that NETs-induced medium increased CD11b expression and decreased the level of the tight-junction protein ZO-1 in vitro.TEER experiments further showed that NETs-induced medium dramatically jeopardized pericytes integrity.All the effects mediated by NETs-induced medium were inhibited by Cl-Amidine,which is a NETs formation inhibitor.To investigate which component of NETs-induced medium drove CD11b induction in pericytes,we collected the cultured medium for proteomics and metabolomics analysis.In PMA-stimulated groups,101 up-regulated and 3 down-regulated proteins were identified.Of note,histones accounted for 30%of the top up-regulated proteins.We selected histones,lactoferrin,N-acetyl-L-cysteine and L-isoleucine to determine whether they change CD11b expression in vitro.The results indicated that histones increased the CD11b m RNA levels and decreased ZO-1 level,while others had no effect on CD11b induction in pericytes.These data reveal that NETs formation significantly increased after TBI,which induced the expression of CD11b in pericytes and decreased the ZO-1 level,and histones were the main contributors for these effects.Section 3:The molecular mechanism underlying the effects of histones within NETs on brain pericytesAfter defining histones as the main components of NETs to affect pericytes,we aimed to investigate the mechanisms of histones on pericytes.According to previous studies,C-type lectin receptors(CLRs)are important receptors for histones recognition.Based on the human protein atlas database(https://www.proteinatlas.org),the expression of Dectin-1(a subtype receptor of CLRs)was relatively high in central nerve system.Our data demonstrated that Dectin-1 expression was positively associated with CD11b upregulation in brain pericytes.According to promoter region sequence analysis of murine CD11b gene,we screened out that c-Jun was possible and important transcription factor for modulating CD11b expression,which was activated by the Dectin-1 receptor.Thus we speculate that the effect of histones to induce CD11b expression may be mediated via Dectin-1 receptors on pericytes surface.Following results showed that the protein expression and glycosylation level of Dectin-1 was markedly upregulated in MBVP treated with NETs-induced medium or histones,while the upregulation of both Dectin-1 and CD11b induced by either NETs-induced medium or histones were attenuated after Dectin-1 was blocked.TEER experiments also confirmed that blocking Dectin-1 was beneficial for restoring pericytes integrity in vitro.From the literature,we discerned that protein kinase Cζ/λ(PKCζ/λ)and the transcriptional factor c-Jun were possible molecules downstream of Dectin-1 activation.The levels of phosphorylated PKC(p-PKCζ/λ)and phosphorylated c-Jun(p-c-Jun)were significantly increased following treatment with NETs-induced medium or histones.Dectin-1 antagonist/si RNAs or PKC inhibitors dramatically decreased the phosphorylation levels of PKCζ/λand c-Jun,along with CD11b downregulation.The luciferase reporter assays and Ch IP experiments further showed that c-Jun could bind to these two CD11b promoter regions(-1250 to-1244 and-410 to-403)directly after stimulation with NETs-induced medium or histones,leading to CD11b induction subsequently.These data prove that NETs-induced medium or histones induced CD11b upregulation in a Dectin-1/PKCζ/λ/c-Jun dependent manner.Section 4:Tentative therapeutic intervention for TBI targeting the mechanism of NETs effect on brain pericytes inductionIn this section,we mainly explored the therapeutic intervention for the restoration of neurological function and BBB integrity of mice after TBI.Immediately after the impact,four groups of TBI mice were administered saline,Cl-Amidine(50mg/kg,NETs inhibitor),Laminarin(25mg/kg,Dectin-1 antagonist)or Laminarin(50mg/kg)via intraperitoneal injection respectively.The results showed that administration of Cl-Amidine and Laminarin significantly reduced the BBB permeability,neutrophils infiltration and death rate during the acute phase after TBI insult.The results of foot-fault test and open field test also supported that Cl-Amidine and Laminarin improved motor and neurological functions in TBI mice after two or four weeks.The data in this section provide evidence that targeting NETs formation and Dectin-1effectively ameliorated BBB disruption and facilitated neurological recovery after TBI,which suggested that NETs and Dectin-1 may become potential targets for treating TBI.