| Objective: Neuropsychiatric systemic lupus erythematosus(NPSLE)is referred as systemic lupus erythematosus(SLE)patients who develop neuropsychiatric symptoms.Among them,brain involvement is called lupus encephalopathy and is a key factor in the poor prognosis of SLE.So far the etiology of lupus encephalopathy remains unclear.Microglia is the resident macrophages in the central nervous system(CNS).Activated microglia accentuates neuroinflammation by secreting proinflammatory mediators and plays an important pathogenic role in a variety of neuroinflammatory and neurodegenerative diseases.Reports have suggested that circulatory inflammatory factors such as complement,cytokines and immune complex(IC)are closely related to the pathogenesis of lupus encephalopathy,but their mechanism is not yet clear.Most of the researches focus on the effects of inflammatory cytokines and autoantibodies/ICs on neurons.However,no evidence has shown the involvement of microglia in lupus encephalopathy.Therefore,we investigate whether inflammatory mediators in peripheral blood of SLE patients can induce microglial activation in vitro to reveal the mechanism of microglial activation in the pathogenesis of lupus encephalopathy.We used a mouse microglial cell line(BV2)in this study because BV2 cells have functions similar to primary microglia.Our research is divided into the following sections:PART1: The efffect of systemic lupus erythematosus serum on microglial activationObjective: To investigate whether serum of SLE patients could activate microglia.Methods: Microglia BV2 cells were subjected to 5% of serum from SLE patients or the healthy controls(HC).The cell viability was detected by MTT and trypan blue assay.The expression of MHC II,CD86 and i NOS protein were detected by western blot.Griess reaction was performed to examine the prodcution of NO.The expression of cytokines(IL-1β,IL-6,TNF-α,IL-4 and IL-10)in the cellular supernatants were investigated by ELISA.Results:1.5% SLE serum(SLE)was not cytotoxic to BV2 microglia.2.SLE caused BV2 microglia amoebiod morphological activation.3.Expression of MHC II and CD86 were significantly upregulated in SLE group.4.SLE caused an obvious increase in i NOS protein expression and subsequent NO release.5.SLE stimulated BV2 microglia to secrete pro-inflammatory cytokines IL-1β,IL-6,TNF-α,but did not affect the secretion of IL-4,IL-10.Conclusion: SLE serum was able to induce the activation of BV2 microglia.PART2: Identification of the components in SLE patients serum that activates microgliaObjective: To clarify the core component in SLE patients serum that activates microglia,we investigated the effect of complement,cytokines(IL-6,TNF-α)and immunoglobulin G-immune complex(Ig G-IC)in the serum of SLE patients on BV2 microglial activation.Methods:a.The effect of complement and cytokines in SLE serum on BV2 microglial activation:1.SLE serum were processed as follows:1)Heat inactivated complement in serum;2)Blocking the effect of cytokine TNF-α by TNF-α neutralizing antibody;3)Blocking the effect of cytokine IL-6 by IL-6 neutralizing antibody;2.The BV2 cells were stimulated by untreated SLE serum and treated SLE serum respectively to detect the differences of MHC II,CD86 and i NOS protein expression,NO production and secretion of cytokines(IL-1β,IL-6 and TNF-α).b.The efffect of SLE Ig G-IC on BV2 microglial activation:1.Experimental groups: Serum of SLE and HC were divided into Ig G-IC(+)and Ig G-IC(-)by Protein G affinity chromatography.1)HC Ig G-IC(+);2)HC Ig G-IC(-):HC serum depletion of Ig G-IC;3)SLE Ig G-IC(+);4)SLE Ig G-IC(-): SLE serum depletion of IgG-IC;2.BV2 cells were exposed to HC Ig G-IC(+),HC Ig G-IC(-),SLE Ig G-IC(+)and SLE Ig G-IC(-)respectively and the differences of MHC II,CD86 and i NOS protein expression,NO production and secretion of cytokines(IL-1β,IL-6 and TNF-α)were assayed between different groups.3.Immunofluorescence was performed to test whether SLE serum components could bind with BV2 microglia.Results:a.SLE serum depletion of complement,TNF-α and IL-6 did not activate BV2 microglia.b.BV2 Microglial activation induced by SLE serum depended on Ig G-IC:1.The up-regulation of MHC II,CD86,i NOS,the production of NO,and the secretion of IL-1β,IL-6,TNF-α were confirmed in SLE Ig G-IC(+)group rather than HC Ig G-IC(+),HC Ig G-IC(-)and SLE Ig G-IC(-).2.Only SLE Ig G-IC could bind to the BV2 microglial cell membrane.Conclusion: BV2 microglial activation induced by SLE serum depended on Ig G-IC rather than complement or cytokines.PART3: Signal transduction mechanisms of BV2 microglial activation induced by SLE IgG-ICObjective: To investigate the signal transduction pathway of BV2 microglial activation induced by SLE Ig G-IC.Methods:1.BV2 microglia were treated with HC Ig G-IC(+),HC IgG-IC(-),SLE Ig G-IC(+)and SLE Ig G-IC(-)respectively.Western blot was used to assay the protein level of TLR9.Additionally,the expression of total and phosphoralated IκBα and NF-κB p65 protien were detected to confirm the activation of NF-κB signaling pathway.2.TLR9 si RNA was used to silence TLR9 expression in BV2 microglia,and the effects of si RNA on NF-κB signaling pathway and microglial activation induced by SLE Ig G-IC were examined.Results:1.SLE Ig G-IC promoted TLR9 protein expression,IκBα protein degradation,IκBαphosphorylation and p65 phosphorylation.2.TLR9 si RNA could inhibit the activation of NF-κB signaling pathway and subsequently reduced the production of NO and secretion of cytokines(IL-1β,IL-6and TNF-α).Conclusion: Ig G-IC in SLE serum could activate proinflammatory phenotype of BV2 microglia through TLR9 /NF-κB signaling pathway. |
