The Role And Mechanism Of Microglia In The Cognitive Impairments Induced By Systemic Lupus Erythematosus Autoantibodies

Objective: Systemic lupus erythematosus(systemic lupus erythematosus,SLE)is a chronic autoimmune disease characterized by multisystemic involvement presenting with numerous clinical manifestations.Proteinuria,hematuria and renal failure are common in renal involvement,ericarditis,endocarditis and coronary artery damage often occurs in the cardiovascular system.When the patients with SLE appear asymptomatic encephalitis,cerebrovascular disease,headache,acute confusion,cognitive decline,emotional disorders and mental illness and other central nervous system(central nervous system,CNS)involvement symptoms,it is called neuropsychiatric systemic lupus erythematosus(neuropsychiatric SLE,NPSLE).The pathological mechanism of NPSLE is still unclear.Autoantibodies against ribosomal P proteins(autoantibodies against ribosomal P protein,anti-P)are strongly associated with the neuropsychiatric manifestations of NPSLE.Animal experiments have shown that anti-P antibodies can impairs spatial memory by attacking hippocampus neurons.However,most of the previous studies on NPSLE have only examined the direct effect of autoantibodies on brain neurons,and microglial cells that play an immune and neuromodulator role in the CNS have received little attention.Moreover,accumulating evidence suggest that activation of microglia in CNS plays an important role in the inflammatory responses in neurological diseases.In order to further explore the correlation between microglia and NPSLE,we conducted a series of in vivo experiments using normal mice as a model to systematically study the effects of autoantibodies from SLE patients on the cytology,electrophysiology,and behavioral functions of mouse brain tissues.To determine whether anti-P Ig G(immunoglobulin G,IgG)can cause abnormal brain electrical activity in mice,and the possible cellular immune mechanism.Exploring the occurrence and development of inflammatory reaction mediated by microglia and whether the application of mTOR(mammalian target of rapamycin)inhibitor can alleviate the social cognitive dysfunction induced by anti-P antibody.So as to achieve the purpose of treating the neural dysfunction induced by anti-P IgG.Methods: 1.We performed intracerebroventricular(intracerebroventricular,ICV)injection of serums collected from SLE patients or healthy controls to mice.Intravital microscopy was used to observe the adherence and adhesion of leukocytes in cerebral microvessels to evaluate the degree of inflammation in the microvessels.2.The injection of complete Freund’s Adjuvant(CFA)containing heat-inactivated H37 Ra mycobacterium tuberculosis and pertussis toxin(PTx)improves the communication of blood brain barrier(BBB)in mice permeability.Then,anti-P IgG from SLE patients was injected via the tail vein to enable it to enter the CNS through BBB.3.A recording electrode was implanted in the primary auditory cortex(A1)of mice.The electroencephalogram(EEG)signal of A1 response to 40-Hz click-train stimulus was recorded to evaluate the auditory steady-state response(ASSR).4.Slice making of brain tissue.Neuron-specific nuclear protein(NeuN),ionic calcium binding adapter molecule(Iba-1)and glial fibrillary acidic protein(GFAP)were used to stain neurons,astrocytes and microglia to evaluate the density and morphology of different types of cells.The presence of apoptosis in the brain tissues was studied using TUNEL assays.The expression of chemokines(CCL2 and CCL5)and adhesion molecules [P-selectin and intercellular adhesion molecule-1(ICAM-1)] were detected.5.PLX3397,an inhibitor of colony-stimulating factor-1 receptor(CSF1R),was used to selectively eliminate the microglia in the brain of mice.Observe the effect of anti-P IgG on mouse brain nerve activity in the absence of microglia.6.Affinity-purified human anti-P antibodies or control IgG(immunoglobulin G)were bilaterally injected into the ventral CA1(vCA1)region of hippocampus in normal mice.Forty-eight hours after IgG administration,social discrimination test was performed to evaluate social memory.Olfactory ability and gross locomotor function were also testified.7.qRT-PCR was used to measure levels of cytokines(tumor necrosis factor-α,interleukin-1,IL-6,interferon-γ),chemokines(CCL2 and CCL5)and neurotrophic factors(IL-4 and IL-10)in the brain tissues.8.To explore whether pretreatment of mice pre-treated with minocycline and rapamycin(rapamycin,Rapa)can alleviate the abnormal performance of the above histological,electrophysiological and behavioral tests.Results:1.Using intravital microscopy,we demonstrated that SLE serum promotes leukocyte rolling and adhesion.We found that ICV injection of SLE serum increases morphological activation of microglia in the cortex and hippocampus.Inflammatory mediators including cytokines,chemokine and adhesion molecules were significantly elevated in the brains of SLE-serum-treated mice.Suppression of microglia activation by systemically using minocycline could decrease the levels of inflammatory molecular,and prevent leukocyte rolling and adhesion.2.Circulating anti-P antibodies caused an enhancement of the ASSR and activation of the microglia through the disrupted BBB,while no obvious neural apoptosis was observed.In contrast,when the microglia were depleted,anti-P antibodies induced a serious reduction in the ASSR and obvious neural apoptosis.3.We first demonstrated that intra-hippocampus injection of anti-P antibodies impairs social recognition in mice,while the olfactory ability and gross locomotor function are not perturbed.The behavioral alterations were accompanied with neuronal cell death,activation of astrocytes and microglia,the production of interleukin(IL-1β,IL-6)and TNF-α increased,and the production of IL-4 and IL-10 decreased in vCA1.The Rapamycin prevented the social recognition impairment induced by anti-P antibodies,and reduced the neurodegeneration and prevented the activation of astrocyte and microglia induced by anti-P.Rapamycin also reduced the production of pro-inflammatory cytokines,and increased the production of anti-inflammatory cytokines.Conclusion: 1.Our date suggests that serum-derived pathogenic factors in SLE patients may cause activation of microglia and adhesion of leukocytes to brain microvessels and other inflammatory reactions.This may be an important reason that BBB function is impaired and autoantibodies in peripheral circulation enter the CNS in large quantities in SLE patients 2.Anti-P IgG from SLE patients can cause abnormal brain electrical activity such as ASSR,which can be restored in the presence of microglia,but irreversible nerve damage will occur if the protection of microglia is lost.3.Microglia mediated neuroinflammatory response may be involved in the anti-P IgG-induced social cognitive behavioral disorder.Rapa can play a protective role in the neural function by inhibiting the neuroinflammatory response.