The Mechanism Of MSCs Reducing Microglia Synaptic Pruning Through CCL8 In The Treatment Of NPSLE

Background:Systemic lupus erythematosus（SLE）is an autoimmune disease involving multiple systems,characterized by lymphocyte hyperactivity,autoantibody production,immune complex deposition,and increased inflammatory cytokines.More than 75%of SLE patients have central nervous system involvements,termed neuropsychiatric SLE（NPSLE）.Recent studies have shown that NPSLE often occurs in the early stage of SLE,suggesting the existence of early-onset pathogenic factors,but the specific mechanism is still unclear,which restricts the development of NPSLE therapeutics.So far,a considerable number of NPSLE patients have unsatisfactory therapeutic effect with conventional treatment.Mesenchymal stem cells（MSCs）have been used in the treatment of many autoimmune diseases due to their low immunogenicity,multi-differentiation potential and immunomodulatory function.There have been reports on the experimental and clinical studies of MSCs in the treatment of SLE,but few studies on NPSLE.Our previous work confirmed that MSCs regulate IFNγsecretion by CD8⁺T cells in the treatment of refractory SLE.This study further explored the effect and mechanism of MSCs on NPSLE,and provided the evidence for MSCs clinical application.Objective:The aim of this study was to investigate the effect and mechanism of MSCs on CD4⁺T cells secreting IFNγ,by which regulated neuronal IFNGR/JAK/STAT/CCL8 signal and microglial synaptic pruning activity in NPSLE,so as to provide the evidence for the clinical application of MSCs.Methods:The behavior of female MRL/lpr and MRL/MPJ mice was analyzed to identify the primary NPSLE model.Six-week-old female C57BL/6 mice were intraperitoneally injected with pristane to induce SLE,the behavioral analysis of mental injury was done ten-week-old.Five-week-old female MRL/lpr mice and ten-week-old pristane induced mice were randomly divided into MSCs transplantation group and PBS treatment group.The MSCs transplantation group was injected with 5×10⁵ cells via tail vein,and the control group was given the same volume of PBS.After 3 weeks and 6 weeks of treatment,the depression-like behavior of mice was detected by ethology to evaluate the therapeutic effect of MSCs.The hippocampal tissue was isolated and stained with HE,immunofluorescence and Golgi staining.Pathology and electrophysiology were used to analyze the neuropathological changes in the brain of mice.Five-week-old MRL/lpr mice were randomly divided into MSCs transplantation group and PBS group.Three weeks after treatment,the mice were killed.RNA sequencing was used to analyze the differentially expressed genes in hippocampus.RNA was extracted and q PCR was used to evaluate microglia phagocytosis and neuron CCL8 expression.The effects of MSCs on CCL8 secretion,microglia activation and synaptic phagocytosis in the brain of MRL/lpr mice were analyzed by immunohistochemistry,immunofluorescence,STED confocal and ELISA.Lupus was induced by intraperitoneal injection of pristane in Ccl8^+/+and Ccl8^-/-mice.The depression-like behavior of the mice after ten-week-old was detected.The mice were killed,and the hippocampus was isolated for neuropathological analysis to evaluate the effect of CCL8 in the pathogenesis of NPSLE.The effect of MSCs on IFNGR/JAK/STAT/CCL8 signal in the hippocampus of MRL/lpr mice treated with MSCs and PBS was verified by q PCR and immunohistochemistry.The primary neurons of Ccl8^+/+and Ccl8^-/-mice were cultured in vitro,and co-cultured with microglia.CCL8 neutralizing antibody or recombinant CCL8 was given,and the phagocytosis of microglia on neuronal synapses was analyzed by confocal analysis.The ratio of CD4⁺IFNγ⁺T cells in peripheral blood or lymph node mononuclear cell was tested by flow cytometry.The effect of MSCs on the secretion of IFNγby CD4⁺T cells was detected by q PCR and ELISA.Pristane induced mice and MRL/lpr mice were treated with STAT1 inhibitor and IFNGR neutralizing antibody（or homotypic control antibody）respectively.After 3 weeks,depressive-like behavior of mice was tested.The mice were killed and hippocampus tissue was isolated for immunohistochemical staining and biological analysis.Results:MRL/lpr mice and pristane induced lupus mice spontaneously developed neuropsychological damage in the early stage of the disease,with depressive-like behavior as the main phenotype.The depressive-like behavior of MRL/lpr mice in MSCs transplantation group was improved,and microglia activation and phagocytosis of synapses were decreased.RNA sequencing and RT-PCR showed that the expression of CCL8 in hippocampus of NPSLE mice was significantly increased,and MSCs treatment inhibited the up-regulation of CCL8.CCL8knockout improved pristane induced depression-like behavior,significantly inhibited microglia phagocytosis and neuron synaptic loss in NPSLE mice.After 3weeks of MSCs transplantation,the ratio of CD4⁺IFNγ⁺T cells in MRL/lpr mice decreased significantly.MSCs could significantly inhibit IFNγexpression in CD4⁺T cells of MRL/lpr mice in vitro.Primary neuron and microglia co-culture experiments showed that IFNγpromoted JAK/STAT1 signal activation,increased CCL8 secretion,induced microglia activation and increased phagocytosis of synapses.MSCs alleviated phagocytosis of synapses by inhibiting IFNγsignal.Intraperitoneal injection of STAT1 inhibitor and IFNGR neutralizing antibody inhibited synaptic loss and alleviated nerve injury in NPSLE mice.Conclusions:MSCs inhibited the IFNγsecretion of CD4⁺T cells,regulated IFNGR/JAK/STAT/CCL8 signal of neurons,reduced the synaptic phagocytosis of microglia to alleviate synaptic loss and relieved NPSLE.