Mechanism Of Chronic Stress-Induced Negative Emotional Behaviors Mediated By Extracellular Nucleosomes

Background Damage-associated molecular patterns(DAMPs)are the primary promoter of progressive neuroinflammation and are associated with chronic stressrelated emotional disorders.The present study investigated the role and mechanism of extracellular nucleosomes and histones,the newly defined DAMPs,in mice with chronic stress.Methods Male C57BL/6 mice were exposed to chronic unpredictable mild stress(CUMS)and corticosterone drinking,respectively,for 4 w.Negative emotional behaviors,including anxiety,depression,cognitive dysfunction,and social interaction disorder were comprehensively investigated.Microglia in bilateral medial prefrontal cortex(mPFC)were harvested by flow cytometer and cell sorting(FACS),and their transcriptomics,morphology,oxidative stress,inflammation,C-type lectin receptor 2D(Clec2d)and Toll-like receptor 9(TLR9)expression,as well as spine morphology were assessed.Spatial expression of citrullination histone H3(citH3)and peptidylarginine deiminase 4(PAD4)were investigated and quantified by immunofluorescence staining and western blot.Extracellular histones and nucluosomes in cerebrospinal fluid(CSF)and interleukin-1β(IL-1β)in mPFC were qualified and quantified by western blot and enzyme linked immune sorbent assay(ELISA),as well as their correlations were investigated by correlation analysis.Specifically,the temporal and concentrationdependent effect of recombinant nucleosomes and histones on viability and(or)proinflammatory ablity in primary neuron and microglia were evaluated by flow cytometry and ELISA in vitro.Besides,microglial pro-inflammatory activation and inflammation were further investigated with stereotactic injection of recombinant nucleosomes and histones in mPFC,and further evaluated with AAV-Clec2d knocking-down,DNaseI,and activated protein C(APC)pre-treatment in vivo.Moreover,the anti-inflammatory effect of knocking-down Clec2d and(or)incubating with TLR9 antagonist E6446 dihydrochloride was further investigated in bone marrow-derived macrophage(BMDM)in vitro.Finally,the rescue effect by AAV-Clec2d knocking-down on microglial oxidative stress and inflammation,together with spine elimination in mPFC and negative emotional behaviors was observed in mice with chronic stress in vivo.Results Mice with chronic stress were presented as obviously anxiety,depression,cognitive dysfunction,and social interaction disorder,and accompanied with significant microglial oxidative stress and inflammation,indicating by reactive oxygen species(ROS)production,primed nuclear factor-κB(NF-κB)signaling pathway,activated NACHT,LRR and PYD domains-containing protein 3(NLRP3)inflammasome,and up-regulated Clec2d and TLR9 in mPFC,together with histones dictation in CSF.The concentration of extracellular nucleosomes in CUMS and CORT mice was positively correlated with IL-1β in mPFC(CUMS vs.CONT:r2=0.8957;CORT vs.CONT:r2=0.8234).Recombinant nucleosomes showed pro-inflammatory ablity on primary microglia,while recombinant histones showed pro-death on primary neuron in a temporal and concentration-dependent manner in vitro.Stereotactic injection of nucleosomes was contributed to promoted microglial inflammation rather than histones in mPFC,indicating the pro-inflammatory role was derived from extracellular histones-bound DNA but not freely histones.AAV-Clec2d knocking-down,DNaseI,and APC were all effective to inhibit nucleosomes-induced microglial oxidative stress and inflammation in vivo.Moreover,the anti-inflammatory effect of knocking-down Clec2d combined with incubating with E6446 dihydrochloride was superior to knocking-down Clec2d alone in BMDM in vitro.AAV-Clec2d knockingdown in mice with chronic stress exhibited reduced microglial oxidative stress and inflammation,together with spine elimination in mPFC and improved negative emotional behaviors in vivo.Conclusion Our findings reveal a novel mechanism of DAMPs-associated inflammation that extracellular nucleosomes accelerates microglial inflammation via Clec2d and TLR9-deprendent NF-κB-NLRP3 inflammasome activation,and then contributes to spine elimination in mPFC and chronic stress-induced negative emotional disorders.